CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. application Ser. No. 17/380,982, filed on Jul. 20, 2021, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/054,228, filed on Jul. 20, 2020. Each of the above-identified applications are hereby incorporated by reference herein in their entireties for all purposes.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically as a text file in ASCII format and is hereby incorporated by reference in its entirety. The name of the ASCII text file is “20-1076-US-CIP_Sequence-Listing_ST25_FINAL.txt.”
FIELD OF THE DISCLOSURE The disclosure relates to Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure also relates to suitable amino acid positions in MMLV RTase for mutagenesis and methods for using MMLV RTase mutants to synthesize cDNA from RNA templates.
BACKGROUND Reverse transcriptase (RTase) enzymes have revolutionized molecular biology. RTase is a critical component of the reverse transcription polymerase chain reaction (RT-PCR) allowing the production of complementary DNA (cDNA) from RNA. The cDNA produced in reverse transcription reactions can be used in a wide range of downstream applications, including quantitative PCR, gene expression analysis, isolated RNA sequencing, gene cloning, and cDNA library creation.
RTases, first derived from retroviruses, facilitate the reverse transcription of RNA into cDNA by utilizing RNA-dependent polymerase and RNase H, a non-sequence-specific endonuclease enzyme that catalyzes cleavage of RNA in an RNA/DNA duplex. This results in virus replication and integration of the viral sequence into host DNA thereby allowing for the proliferation of the virus along with host DNA. Within the laboratory setting, RTases from Moloney murine leukemia virus (MMLV), avian myeloblastosis virus (AMV), and human immunodeficiency virus type 1 (HIV-1) are the most commonly used RTase for cDNA synthesis.
RTases for research applications are often mutated multi-generational MMLV and AMV RTases that have been optimized for laboratory procedures. Mutations in the RTases alter properties of the enzymes, including thermostability, RTase activity, 5′ mRNA coverage, and RNase H activity.
AMV RTases are thermostable and less sensitive to thermal degradation than MMLV RTase and are preferred for RNA having a strong secondary structure. In addition, AMV RTases are often suitable for use with RNA molecules that are five kilobases or longer because of the heat stability of AMV RTases. However, the high temperatures required to resolve strong secondary structures or long RNA strands can negatively impact RNA integrity and fidelity of transcription. AMV also possess an intrinsic RNase activity that degrades RNA in an RNA/DNA hybrid, which can result in reduced total cDNA and reduced full-length cDNA yield.
MMLV RTase is characterized by low RNase H activity and a higher fidelity as compared to AMV RTase. The reduced RNase H activity allows MMLV RTases to be used for the reverse transcription of long RNAs (>5 kb). However, the RNase H activity of MMLV RTase limits the efficiency of synthesizing long cDNA in vitro. Mutations in MMLV RTase have been introduced to reduce RNase H activity. In addition, because the optimal temperature for MMLV RTase activity is ˜37° C., the enzyme lacks the ability to effectively reverse transcribe RNAs with strong secondary structures. The use of MMLV RTase at elevated temperatures can compromise cDNA length and yield as a result of lower enzyme activity. MMLV RTase mutants that substitute Mn2+ for Mg2+ in the reaction mixture attempt to overcome these limitations, but are characterized by inefficiency and error.
Thus, despite the unique properties of AMV and MMLV RTases, there exists a need for an RTase that combines the beneficial attributes of AMV and MMLV RTases. Consistent with this, the present application discloses MMLV RTase mutants, isolated through rational mutagenesis of MMLV RTase, that exhibit increased RTase activity and thermostability as compared to RTases, including RNase H minus constructs, that are currently available in the art.
SUMMARY The disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure also provides suitable amino acid positions in MMLV RTase for mutagenesis and methods and kits for using MMLV RTase mutants to synthesize cDNA from RNA templates.
One aspect of the disclosure provides an isolated Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutant comprising the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least two amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) threonine to lysine at position 306 (T306K); (i) a valine to asparagine at position 433 (V433N); (j) a valine to arginine at position 433 (V433R); (k) an isoleucine to glutamic acid at position 593 (1593E); or (1) an isoleucine to tryptophan at position 593 (I593W).
Another aspect of the disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an MMLV RTase mutant of the disclosure.
Other aspects of the disclosure provide a composition or a kit comprising an MMLV RTase mutant of the disclosure.
Other aspects of the disclosure provide methods for synthesizing complementary deoxyribonucleic acid (cDNA) or methods for performing reverse transcription-polymerase chain reaction (RT-PCR) using an MMLV RTase mutant of the disclosure.
Specific embodiments of the disclosure will become evident from the following more detailed description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1C are schematics showing reverse transcriptase mutagenesis selection by rational design. Amino acid positions for mutagenesis were chosen at the substrate binding site (FIGS. 1A and 1B) or near the substrate binding site (FIG. 1C).
FIG. 2 shows Western blot analysis of test induction results in in BL21(DE3) cells for MMLV RT in TB medium. Lane 1—Precision Plus Protein Unstained Standards (Bio Rad, Cat #161-0363), Lane 2—Time=0 hour, Lane 3—Time=3 hours after induction at 37° C., Lane 4—Time=0 hour, Lane 5—Time=21 hours after induction at 18° C.
DETAILED DESCRIPTION The disclosure relates to Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure also relates to suitable amino acid positions in MMLV RTase for mutagenesis and methods and kits for using MMLV RTase mutants to synthesize cDNA from RNA templates.
The MMLV RTase mutants of the disclosure, which have been identified and isolated, at least in part, through rational mutagenesis of a base construct of MMLV RTase, were found to have increased RTase activity and thermostability as compared to wild-type MMLV RTase and certain MMLV RTase mutants, including RNase H minus RTases, that are currently available in the art.
Reference will now be made in detail to exemplary embodiments of the claimed invention. While the claimed invention will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the claimed invention to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents, as may be included within the spirit and scope of the claimed invention, as defined by the appended claims.
Those of ordinary skill in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the claimed invention. In addition, although certain methods and materials are described herein, other methods and materials that are similar or equivalent to those described herein can also be used to practice the claimed invention.
In addition, any of the compositions or methods provided, disclosed, or described herein can be combined with one or more of any of the other compositions and methods provided, disclosed, or described herein.
1. Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the claimed invention belongs. The terminology used herein is for describing particular embodiments only and is not intended to be limiting of the claimed invention. All technical and scientific terms used herein have the same meaning.
The following references provide those of skill in the art with a general understanding of many of the terms used herein (unless defined otherwise herein): Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd ed. (Wiley, 2006); Walker, The Cambridge Dictionary of Science and Technology (Cambridge University Press, 1990); Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed. (Springer Verlag, 1991); and Hale et al., Harper Collins Dictionary of Biology (HarperCollins Publishers, 1991). Generally, the procedures or methods described herein and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as, for example, Green et al., Molecular Cloning: A Laboratory Manual, 4th ed. (Cold Spring Harbor Laboratory Press, 2012), and Ausubel, Current Protocols in Molecular Biology (John Wiley & Sons Inc., 2004).
The following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings known or understood by those having ordinary skill in the art are also possible, and within the scope of the claimed invention. All publications, patent applications, patents, and other references mentioned or discussed herein are expressly incorporated by reference in their entireties. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
As used herein, the singular forms “a,” “and,” and “the” include plural references, unless the context clearly dictates otherwise.
As used herein, the term “or” means, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
As used herein, the term “including” means, and is used interchangeably with, the phrase “including but not limited to.”
As used herein, the term “such as” means, and is used interchangeably with, the phrase “such as, for example” or “such as but not limited.”
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example, within two standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms “nucleic acid molecule” and “polynucleotide” refer to a polymer or large biomolecule comprised of nucleotides. The term “nucleic acid” includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogs thereof. Non-limiting examples of nucleic acid molecules include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA, rRNA, cRNA, tRNA), and chimeras thereof. A nucleic acid molecule can be obtained by cloning techniques or synthesized, using techniques that are known to those of skill in the art. DNA can be double-stranded or single-stranded (coding strand or non-coding strand, i.e., antisense). A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as “peptide nucleic acids” (PNA)), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, for example, 2′ methoxy substitutions (containing a 2′-O-methylribofuranosyl moiety) and/or 2′ halide substitutions. Nitrogenous bases may be conventional bases (adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U)), known analogs thereof (e.g., inosine), known derivatives of purine or pyrimidine bases, or “abasic” residues in which the backbone includes no nitrogenous base for one or more residues. A nucleic acid may comprise only conventional sugars, bases, and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs). An “isolated nucleic acid molecule,” as is generally understood by those of skill in the art and as used herein, refers to a polymer of nucleotides, and includes but is not limited to DNA and RNA.
As used herein, the term “probe” refers to a nucleic acid oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence). A probe's “target” generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or “base pairing.” Sequences that are “sufficiently complementary” allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary. A probe may be labeled or unlabeled. A probe can be produced by molecular cloning of a specific DNA sequence or it can be synthesized. Probes for use in the methods disclosed herein can be readily designed and used by those of skill in the art.
As used herein, the term “primer” refers to a nucleic acid oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid or its complement, and which is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Primers may be provided in double-stranded or single-stranded form. Primers for use in the methods disclosed herein can be readily designed and used by those of skill in the art.
Probes or primers for use in the methods disclosed herein may be of any suitable length, depending on the particular assay format and the particular needs and targeted sequences employed. For example, the probes or primers for use in the methods disclosed herein are at least 10 nucleotides in length, or at least 15, 20, 25, 30, or more than 30 nucleotides in length, and they may be adapted to be especially suited for a chosen nucleic acid amplification system and/or hybridization system used. Longer probes and primers are also within the scope of the disclosure.
A “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analog of such RNA or cDNA) that is complementary to or having a high percentage of identity (e.g., at least 80% identity) with all or a portion of a mature mRNA made by transcription of a marker of the disclosure and normal post-transcriptional processing (e.g., splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
As used herein, the terms “reverse transcriptase,” “RTase,” or “RT” refer to an enzyme that is used to generate complementary (cDNA) from an RNA template in a process known as “reverse transcription.” The term reverse transcriptase, as used herein, also refers to any enzyme that exhibits reverse transcription activity. Reverse transcriptases can be derived from a variety of sources including but not limited to viruses including retroviruses and DNA polymerases exhibiting transcriptase activity. Such retroviruses include but are not limited to Moloney murine leukemia virus (MMLV), avian myeloblastosis virus (AMV), and human immunodeficiency virus (HIV).
Reverse transcriptase activity can be measured by incubating an RTase in a buffer containing an RNA template and deoxynucleotides. One of skill in the art will recognize that a wide range of conditions can be used to perform reverse transcription reactions and multiple methods exist for measuring the quantity of cDNA produced during reverse transcription.
Reverse transcriptases of the disclosure include reverse transcriptases having one or a combination of the properties described herein. Such properties include but are not limited to increased activity, enhanced DNA synthesis, enhanced stability or enhanced thermostability, reduced or eliminated RNase H activity, reduced terminal deoxynucleotidyl transferase activity, increased accuracy or increased fidelity, increased specificity, or altered half-life, for example when compared to a base construct. As used herein, the term “base construct” refers to the initial RTase from which the RTase mutants of the disclosure are prepared (e.g. for example a wild-type RTase or a modified wild-type RTase).
As used herein, the terms “accuracy” and “fidelity” are used interchangeably and refer to ability of an RTase to accurately replicate a desired template; i.e., the ability of the RTase to accurately perform cDNA synthesis in a reverse transcription reaction. The “fidelity” or “accuracy” of a reverse transcriptase can be assessed by determining the frequency of incorrect nucleotide incorporation into the synthesized cDNA molecule, which may be referred to as the enzyme's error rate. As used herein, the term “increased fidelity” refers to RTase mutants of the disclosure that exhibit an error rate lower than that of the base construct. For example, the RTase mutants as disclosed herein can exhibit an error rate that is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 100%, or 200% lower than, or at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, or more than 10-fold lower than the error rate of the RTase base construct . . . .
As used herein, the term “specificity” refers to a decrease in mis-priming by an RTase during cDNA synthesis. An RTase mutant's specificity can be assessed by performing a reverse transcription reaction at a particular temperature, including higher temperatures, and comparing the amount of mis-priming in that reaction with the amount of mis-priming in a reaction performed with the wild-type RTase (or the RTase base construct) under identical conditions.
As used herein with respect to the RTase molecules of the disclosure, the terms “stable” and “thermostable” are used interchangeably and refer to an enzyme that is resistant to heat inactivation and remains active at temperatures in excess of 37° C. (e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 70° C., or higher temperatures). For example, in one embodiment the disclosure provides an RTase mutant having activity with a longer half-life than that of the base construct RTase at an elevated temperature. Thus, RTase mutants with “enhanced thermostability” can refer to RTase mutants of the disclosure that exhibit an increase in thermostability at temperatures of about 50° C. up to about 90° C. as compared to the base construct RTase. In some embodiments, the thermostability of the RTase mutant is at least 1.5 fold or greater as compared to the thermostability of the base construct RTase. Comparisons of cDNA produced by a base construct and RTase mutant are compared using identical reaction conditions for the base construct and RTase mutant reactions. Reaction conditions can include but are not limited to salt concentration, buffer concentration, pH, divalent metal ion concentration, temperature, nucleoside triphosphate concentration, template concentration, RTase concentration, primer concentration, time, and in one-step PCR, the quantitative PCR primer and probe concentrations.
As used herein, the term “enhanced DNA synthesis” refers to an RTase enzyme that produces more DNA (e.g. cDNA) than the base RTase construct. In some embodiments, DNA synthesis can be measured by quantitative PCR at standard reaction conditions, as compared to the base construct RTase. Consistent with assessments of thermostability, quantitative comparisons are made under similar or the same reaction conditions and the amount of cDNA synthesized using the base construct RTase is compared to the amount of cDNA produced using the RTase mutant (see Tables 4-7). In some embodiments, the RTase mutant of the disclosure with enhanced DNA synthesis may produce about 5% to about 200% more cDNA than the base construct RTase. In some embodiments, the RTase mutant of the disclosure with enhanced DNA synthesis has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or 200% more than, or at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, or more than 10-fold more DNA synthesis than the RTase base construct DNA synthesis.
Reverse transcriptase activity, as described herein, was evaluated in a one-step or two-step procedure. The one-step procedure combines reverse transcription and quantitative PCR in a single reaction. The method is performed by including Gene Expression Master Mix, RTase, RNA, a fluorescent probe, and primers and probes as described in Example 3. The two-step procedure comprises reverse transcription followed by quantitative PCR. In the reverse transcription step, RTase is added to a mixture containing RNA, gene specific primers, first strand synthesis buffer, and RNase. The resultant cDNA is then quantified in a second step wherein the cDNA is combined with Gene Expression Master Mix, primers and probes, and a fluorescent marker. The cDNA produced in either the one-step and two-step procedures is quantified, and the mean and standard deviation reported as shown herein in Tables 4-7.
As used herein, “RNase H activity” refers to cleavage of RNA in DNA-RNA duplexes via a hydrolytic mechanism to produce 5′ phosphate terminated oligonucleotides. RNase H activity does not include degradation of single-stranded nucleic acids, duplex DNA, or double-stranded RNA. As used herein, the phrase “substantially lacks RNase H activity” means having less than 10%, 5%, 1%, 0.5%, or 0.1% of the activity of a wild type enzyme. As used herein, the phrase “lacks RNase H activity” means having undetectable RNase H activity or having less than about 1%, 0.5%, or 0.1% of the RNase H activity of a wild type enzyme.
As used herein, the term “mutation” refers to a change introduced into the nucleic acid sequence encoding a protein that changes the amino acid sequence of the protein, including but not limited to substitutions, insertions, deletions, point mutations, transpositions, inversions, frame shifts, nonsense mutations, truncations, or other forms of aberrations. A mutation may produce no discernible changes or result in a new property, function, or trait of the mutated protein. An RTase mutant of the disclosure may have one or more mutations in the nucleic acid sequence encoding the RTase mutant resulting in one or more mutations in the amino acid sequence of the RTase mutant. A mutation can result in one or more amino acids being substituted for an alternate amino acid residue, including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and/or Val. The resulting amino acid mutations may impart altered functional and biological properties to the RTase mutant including but not limited to increased activity, enhanced DNA synthesis, enhanced stability or enhanced thermostability, reduced or eliminated RNase H activity, reduced terminal deoxynucleotidyl transferase activity, increased accuracy or increased fidelity, increased specificity, or altered half-life.
As used herein, the terms “detecting,” “detection,” “determining,” and the like refer to assays performed for identification of the quantity of cDNA synthesis as a marker of RTase activity. The amount of marker expression or activity detected in the sample can be the same as, decreased, or increased as compared to the amount of marker expression or activity detected using the RTase base construct. One of skill in the art will understand that amount of cDNA can be quantified using multiple techniques.
The term “increased,” as used herein with regard to RTase activity, refers to the level of RTase activity of an RTase mutant as compared to the RTase base construct. An RTase mutant has “increased” RTase activity if the level of its RTase activity, as measured by the quantity of cDNA synthesized or as measured by other methods known in the art, is more than the RTase base construct activity. For example, the RTase activity of the RTase mutant is increased if the RTase activity is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than, or at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, or more than 10-fold more than the RTase base construct activity.
The term “decreased,” as used herein with regard to RTase activity, refers to the level of RTase activity of an RTase mutant as compared to the RTase base construct. An RTase mutant has “decreased” RTase activity if the level of its RTase activity, as measured by the quantity of cDNA synthesized or as measured by other methods known in the art is less than the RTase base construct activity. For example, the RTase activity of the RTase mutant is decreased if the RTase activity is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% less than, or at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more than 10-fold less than the RTase base construct activity.
As used herein, the term “amplification” refers to any known in vitro procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. Known in vitro amplification methods include, for example, transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification, and strand-displacement amplification (SDA, including multiple strand-displacement amplification method (MSDA)). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Q-β-replicase. PCR amplification uses DNA polymerase, primers, and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA. PCR involves denaturation of a double-stranded DNA molecule, followed by annealing of DNA primers directed to the sequence of interest, and amplification/extension of the newly formed DNA strand. LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation. SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps. Other strand-displacement amplification methods known in the art (e.g., MSDA) do not require endonuclease nicking. Those of skill in the art will understand that the oligonucleotide primer sequences of the disclosure may be readily used in any in vitro amplification method based on primer extension by a polymerase. As commonly known in the art, oligonucleotides are designed to bind to a complementary sequence under selected conditions.
As used herein, “real time PCR” or “quantitative PCR” refers to a PCR method wherein the amount of product being formed can be monitored using florescent probes and quantified by tracking the fluorescent signal produced, above a threshold level. Real time PCR can be performed in a one-step reaction that includes the reverse transcription step in a simultaneous reaction (i.e., real time PCR or RT-PCR) or in a two-step reaction in which the reverse transcription step and PCR steps are performed consecutively.
As used herein, the term “complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide of the first region is capable of base pairing with a nucleotide of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotides of the first portion are capable of base pairing with nucleotides in the second portion. In another embodiment, all nucleotides of the first portion are capable of base pairing with nucleotides in the second portion.
Polypeptide and polynucleotide sequences may be aligned, and percentages of identical amino acids or nucleotides in a specified region may be determined against another polypeptide or polynucleotide sequence, using computer algorithms that are publicly available. The percent identity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the disclosure; and then multiplying by 100 to determine the percent identity.
As used herein, the terms “sample” and “biological sample” include a specimen or culture obtained from any source. Biological samples can be obtained from cerebrospinal fluid, lacrimal fluid, blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), urine, saliva, and the like. Biological samples also include tissue samples, such as biopsy tissues or pathological tissues that have previously been fixed (e.g., formaline snap frozen, cytological processing).
2. Reverse Transcriptases The disclosure relates to Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The MMLV RTase mutants of the disclosure are prepared by modifying the sequence of an MMLV RTase base construct (SEQ ID NO: 637). In one embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least one amino acid substitution that is: (a) an isoleucine to arginine, lysine, or methionine substitution at position 61 (I61R, I61K, or I61M); (b) a glutamine to arginine, lysine, or isoleucine substitution at position 68 (Q68R, Q68K, or Q68I); (c) a glutamine to arginine, histidine, or isoleucine substitution at position 79 (Q79R, Q79H, or Q79I); (d) a leucine to arginine, lysine, or asparagine substitution at position 99 (L99R, L99K, or L99N); (e) a glutamic acid to aspartic acid, methionine, or typtophan substitution at position 282 (E282D, E282M, or E282W); and/or (f) an arginine to alanine substitution at position 298 (R298A).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least two amino acid substitutions that are: (a) an isoleucine to arginine substitution at position 61 and a glutamic acid to aspartic acid substitution at position 282 (I61R/E282D); (b) a leucine to arginine at substitution position 99 and a glutamic acid to aspartic acid substitution at position 282 (L99R/E282D); (c) a glutamine to arginine substitution at position 68 and a glutamic acid to aspartic acid substitution at position 282 (Q68R/E282D); (d) a glutamine to arginine substitution at position 79 and a glutamic acid to aspartic acid substitution at position 282 (Q79R/E282D); (e) a glutamic acid to aspartic acid substitution at position 282 and an arginine to alanine substitution at position 298 (E282D/R298A); (f) an isoleucine to arginine substitution at position 61 and a leucine to arginine substitution at position 99 (I61R/L99R); (g) an isoleucine to arginine substitution at position 61 and a glutamine to arginine substitution at position 68 (I61R/Q68R); (h) an isoleucine to arginine substitution at position 61 and a glutamine to arginine substitution at position 79 (I61R/Q79R); (i) an isoleucine to arginine substitution at position 61 and an arginine to alanine substitution at position 298 (I61R/R298A); (j) a glutamine to arginine substitution at position 68 and a leucine to arginine substitution at position 99 (Q68R/L99R); (k) a glutamine to arginine substitution at position 79 and a leucine to arginine substitution at position 99 (Q79R/L99R); (1) a leucine to arginine at substitution position 99 and an arginine to alanine substitution at position 298 (L99R/R298A); (m) a glutamine to arginine substitution at position 68 and a glutamine to arginine substitution at position 79 (Q68R/Q79R); (n) a glutamine to arginine substitution at position 68 and an arginine to alanine substitution at position 298 (Q68R/R298A); or (o) a glutamine to arginine substitution at position 79 and an arginine to alanine substitution at position 298 (Q79R/R298A).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least three amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/L99R/E282D); (b) a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q79R/L99R/E282D); (c) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 68, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/E282D); or (d) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 68, and a leucine to arginine substitution at position 99 (Q68R/Q79R/L99R).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least four amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/L99R/E282D); (b) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to lysine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/L99K/E282D); (c) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to asparagine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79R/L99N/E282D); (d) a glutamine to isoleucine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68I/Q79R/L99R/E282D); (e) a glutamine to lysine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68K/Q79R/L99R/E282D); (f) a glutamine to arginine substitution at position 68, a glutamine to histidine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79H/L99R/E282D); (g) a glutamine to arginine substitution at position 68, a glutamine to isoleucine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (Q68R/Q79I/L99R/E282D); (h) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to methionine substitution at position 282 (Q68R/Q79R/L99R/E282M); (i) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to tryptophan substitution at position 282 (Q68R/Q79R/L99R/E282W); or (j) a glutamine to isoleucine substitution at position 68, a glutamine to histidine substitution at position 79, a leucine to lysine substitution at position 99, and a glutamic acid to methionine substitution at position 282 (Q68I/Q79H/L99K/E282M).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least five amino acid substitutions that are: (a) an isoleucine to lysine substitution at position 61, a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (I61K/Q68R/Q79R/L99R/E282D); (b) an isoleucine to methionine substitution at position 61, a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, and a glutamic acid to aspartic acid substitution at position 282 (I61M/Q68R/Q79R/L99R/E282D); or (c) an isoleucine to methionine substitution at position 61, a glutamine to isoleucine substitution at position 68, a glutamine to histidine substitution at position 79, a leucine to lysine substitution at position 99, and a glutamic acid to methionine substitution at position 282 (I61M/Q68IR/Q79H/L99K/E282M).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 637, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least five or more amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a valine to arginine substitution at position 433, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E): (b) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 82, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a valine to arginine substitution at position 433, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E); (c) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 82, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a threonine to glutamic acid substitution at position 332, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E); (d) a glutamine to arginine substitution at position 68, a glutamine to arginine substitution at position 79, a leucine to arginine substitution at position 82, a leucine to arginine substitution at position 99, a glutamic acid to aspartic acid substitution at position 282, a glutamine to glutamic acid substitution at position 299, a threonine to glutamic acid substitution at position 332, a valine to arginine substitution at position 433, and a isoleucine to glutamic acid at position 593 (Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises at least two amino acid substitutions that are: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) threonine to lysine at position 306 (T306K); (i) a valine to asparagine at position 433 (V433N); (j) a valine to arginine at position 433 (V433R); (k) an isoleucine to glutamic acid at position 593 (I593E); or (1) an isoleucine to tryptophan at position 593 (I593W).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises the amino acid substitutions: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine substitution at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine substitution at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) a threonine to lysine substitution at position 306 (T306K); (i) a valine to asparagine substitution at position 433 (V433N); and (j) an isoleucine to tryptophan substitution at position 593 (I593W).
In another embodiment, the MMLV RTase mutant of the disclosure comprises the amino acid sequence of SEQ ID NO: 717, wherein the amino acid sequence of the MMLV RTase mutant further comprises the amino acid substitutions: (a) a glutamine to arginine substitution at position 68 (Q68R); (b) a glutamine to arginine substitution at position 79 (Q79R); (c) a leucine to tyrosine substitution at position 82 (L82Y); (d) a leucine to arginine substitution at position 99 (L99R); (e) a leucine to isoleucine substitution at position 280 (L280I); (f) a glutamic acid to aspartic acid substitution at position 282 (E282D); (g) a glutamine to glutamic acid substitution at position 299 (Q299E); (h) a threonine to lysine substitution at position 306 (T306K); (i) a valine to arginine substitution at position 433 (V433R); and (j) an isoleucine to glutamic acid substitution at position 593 (I593E).
In one embodiment the RTase mutant amino acid sequence comprises a mutant selected from Tables 3, 8, 9, 12, 21, 22, or 38. In one aspect, the RTase mutant amino acid sequence comprises a mutant selected from the amino acid sequences of SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, SEQ ID NO: 651, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 679, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, SEQ ID NO: 699, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 728, SEQ ID NO: 729, SEQ ID NO: 730, or SEQ ID NO: 731.
In one embodiment the RTase mutant amino acid sequence comprises a C-terminal extension. In one aspect the C-terminal extension comprises a peptide sequence. In another embodiment an isolated polypeptide encodes a RTase mutant with a C-terminal extension
The claimed invention is based, at least in part, on the discovery that certain single and double amino acid mutations introduced into an MMLV RTase sequence, as disclosed herein, result in an MMLV RTase with increased or enhanced thermostability and/or RTase activity. Accordingly, methods for synthesizing the MMLV RTase mutants and methods for performing reverse transcription-polymerase chain reaction (RT-PCR) are also provided herein. Further provided are kits comprising the isolated MMLV RTase single, double, triple, or more mutations.
In certain embodiments, the mutated RTase is derived from the retrovirus Moloney murine leukemia virus (MMLV). In other embodiments, a mutated RTase of the disclosure could be derived from the RTase from a retrovirus other than MMLV, such as avian myeloblastosis virus (AMV) or human immunodeficiency virus type 1 (HIV-1), by introducing the same mutations into an RTase base construct obtained from the other retrovirus.
In certain embodiments, the RTase mutants of the disclosure are obtained by genetic engineering techniques that are well known in the art. For example, site-directed and random mutagenesis can be used to generate the RTase mutants of the disclosure.
In one embodiment of the disclosure, an RTase mutant of the disclosure is part of a composition.
3. Mutagenesis The RTase mutants of the disclosure can be prepared by standard methods disclosed herein or known in the art. In one embodiment, the nucleic acid sequence of the RTase base construct (SEQ ID NO: 637) is modified to create a nucleic acid sequence encoding an RTase mutant. One of skill in the art will recognize that colonies with the appropriate strains can be used to grow and express an RTase mutant of interest, and following cell harvest and protein isolation, the RTase mutant can be used in cDNA synthesis techniques. Non-limiting examples of mutagenesis and cDNA synthesis are described herein in Examples 1-3.
As used herein, the term “mutagenesis” refers to the introduction of a genetic change in the nucleic acid sequence of a cell, wherein the alteration is then inherited by each cell. One of skill in the art will understand that mutations in a given nucleic acid sequence can be introduced using a variety of methods. One of skill in the art will further recognize that mutagenesis methods seek to mutate a target gene or target polynucleotide. The target gene may encode any one or more desired proteins. Mutagenesis methods commonly use a synthetic oligonucleotide that carries the desired sequence modification. The mutagenic oligonucleotide is incorporated into the DNA sequence using in vitro enzymatic DNA synthesis and is propagated in a mutant or wild-type bacterium.
Site directed mutagenesis, wherein targeted mutations are introduced into one or more desired positions of a template polynucleotide, may be achieved using primer extension mutagenesis. This technique requires the use of a specific primer that contains one or more desired mutations relative to the template polynucleotide. The mutagenesis primer can be a synthetic oligonucleotide or a PCR product. The mutated primer may include one or more substitutions, deletions, additions, or combinations thereof.
Mutated reverse transcriptases may also be generated using random mutagenesis, wherein mutations are introduced into the mutagenesis primer during synthesis. Randomly mutagenized oligonucleotides may also be used as mutagenesis primers.
In another embodiment, the mutated reverse transcriptases of the disclosure can be developed using error-prone rolling circle amplification (RCA). In this technique, the fidelity of a DNA polymerase is decreased by performing the RCA in the presence of MnCl2 or by decreasing the amount of input DNA.
4. cDNA Synthesis The disclosure also relates to the activity of MMLV RTases, as measured by the quantity of cDNA produced by the MMLV RTases disclosed herein. cDNA can be prepared using one-step or two-step procedures and can be obtained from a variety of template molecules. As used herein, the term “template molecule” refers to a biological molecule that carries the genetic code for use in making a new nucleic acid strand. For example, in DNA replication, the unwound double helix and each single-stranded DNA molecule is used as a template to synthesize a complementary strand. Reverse transcription generates cDNA from RNA. One of skill in the art will understand that cDNA molecules may be prepared from a variety of nucleic acid template molecules. In one embodiment, the nucleic acid template can be single-stranded or double-stranded DNA. In one embodiment, RNA can be used in cDNA synthesis. In certain embodiments, the MMLV RTase mutants of the disclosure exhibit increased or enhanced thermostability and/or RTase activity as compared to an RTase base construct. In other embodiments, the MMLV RTase mutants of the disclosure exhibit altered half-life, reduced or eliminated RNase H activity, reduced terminal deoxynucleotidyl transferase activity, increased accuracy or fidelity, or increased specificity.
The disclosure also provides methods for synthesizing cDNA using the MMLV RTase mutants of the disclosure that have single or double amino acid mutations. The MMLV RTase mutants of the disclosure may be used in methods that produce a first strand cDNA or a first and second strand cDNA. One of skill in the art will understand that first and second strand cDNA may form a double-stranded DNA molecule, which may include a full-length cDNA sequence and cDNA libraries.
The cDNA molecules that have been reverse transcribed by the MMLV RTase mutants of the disclosure may be isolated, or the reaction mixture containing the cDNA molecules may be directly used in downstream applications or for further analysis or manipulation. Amplification methods that may be used to practice the methods of the disclosure are described herein and are well known in the art. Reverse transcription reactions may be carried out using non-specific primers, such as an anchored oligo-dT primer, or random sequence primers, or using a target-specific primer complementary to the RNA for each genetic probe being monitored, or using thermostable DNA polymerases (such as AMV RTase or MMLV RTase).
Amplification methods utilize pairs of primers that selectively hybridize to nucleic acids corresponding to a specific nucleotide sequence of interest that are contacted with the isolated nucleic acid under conditions that permit selective hybridization. Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced. Next, the amplification product is detected. In certain methods, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label, or even via a system using electrical or thermal impulse signals.
Methods based on ligation of two (or more) oligonucleotides in the presence of a nucleic acid having the sequence of the resulting “di-oligonucleotide,” thereby amplifying the di-oligonucleotide, also may be used in the amplification step of the disclosure.
In some embodiments of the disclosure, the detection process can utilize a hybridization technique, for example, wherein a specific primer or probe is selected to anneal to a target biomarker of interest, and thereafter detection of selective hybridization is made. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence.
One of skill in the art will recognize that cDNA molecules made using the MMLV RTase mutants of the disclosure can be used in a variety of additional downstream applications. For example, amplification methods may include one-step PCR, two-step PCR, real-time or quantitative PCR, hot-start PCR, nested PCR, touch down PCR, differential display PCR (DDRT-PCR), microarray technologies, inverse PCR, Rapid amplification of PCR ends (RACE or anchored PCR), multiplex PCR, and site directed PCR mutagenesis. Synthesized cDNA and cDNA libraries created with the MMLV RTase mutants of the disclosure can be used in cloning and/or sequencing for further characterization. One of skill in the art will recognize that nucleic acid amplification using cDNA prepared with the MMLV RTase mutants of the disclosure may include additional techniques not listed herein.
To enable hybridization to occur under the methods presented above, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of the sequence of interest.
5. Biological Samples The MMLV RTase mutants and associated methods of the disclosure may be practiced with any suitable biological sample from which RNA or DNA can be isolated. In one embodiment of the disclosure, the biological sample may be a bodily fluid or tissue obtained from either a diseased or a healthy subject. In some embodiments of the disclosure, the biological sample may be a bodily fluid, including but not limited to whole blood, plasma, serum, feces, or urine. In another embodiment, the methods of the disclosure may be practiced with any suitable samples that are freshly isolated or that have been frozen or stored after having been collected from a subject, for example, with a known diagnosis, treatment, and/or outcome history. Samples may be collected by any non-invasive means, such as, for example, fine needle aspiration or needle biopsy, or alternatively, by an invasive method, including, for example, surgical biopsy. In such embodiments, RNA or DNA can be extracted from a biological sample (e.g., blood serum) before analysis. Methods of RNA and DNA extraction are well known in the art.
A number of kits for use in extracting RNA (i.e., total RNA or mRNA) from bodily fluids or tissues (e.g., blood serum) and are known in the art and commercially available. One of ordinary skill in the art can easily select an appropriate kit for a particular situation.
In certain embodiments of the disclosure, after extraction, mRNA is amplified, and transcribed into cDNA, which can then serve as template for multiple rounds of transcription by the appropriate RNA polymerase. Amplification methods that may be used to practice the methods of the disclosure are described herein and are well known in the art. Reverse transcription reactions may be carried out using non-specific primers, such as an anchored oligo-dT primer, or random sequence primers, or using a target-specific primer complementary to the RNA for each genetic probe being monitored, or using thermostable DNA polymerases, such as MMLV RTase or the MMLV RTase mutants of the disclosure.
In certain embodiments, the RNA isolated from a biological sample (e.g., after amplification and/or conversion to cDNA or cRNA) is labeled with a detectable agent before being analyzed. The role of a detectable agent is to facilitate detection of RNA or to allow visualization of hybridized nucleic acid fragments (e.g., nucleic acid fragments hybridized to genetic probes in an array-based assay). In some embodiments, the detectable agent is selected such that it generates a signal which can be measured and whose intensity is related to the amount of labeled nucleic acids present in the sample being analyzed.
Methods for labeling nucleic acid molecules are well known in the art. A review of labeling protocols and label detection techniques can be found in Kricka, Ann. Clin. Biochem. 39: 114-29 (2002); van Gijlswijk et al., Expert Rev. Mol. Diagn. 1: 81-91 (2001); and Joos et al., J. Biotechnol. 35: 135-53 (1994). Standard nucleic acid labeling methods include incorporation of radioactive agents; direct attachment of fluorescent dyes or of enzymes; chemical modifications of nucleic acid fragments making them detectable immunochemically or by other affinity reactions; and enzyme-mediated labeling methods, such as random priming, nick translation, PCR, and tailing with terminal transferase.
Any of a wide variety of detectable agents can be used to practice the methods of the disclosure. Suitable detectable agents include but are not limited to various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, and phosphors), enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, and alkaline phosphatase), colorimetric labels, magnetic labels, biotin, digoxigenin, or other haptens and proteins for which antisera or monoclonal antibodies are available.
6. Kits The disclosure also provides kits for use in reverse transcription or related technologies. These kits include one or more of the following: an MMLV RTase mutant enzyme, reagents and buffers for conducting a reverse transcriptase reaction, a box, vial tubes, ampules, and the like. Kits can also include instructions for use of the kit for practicing any of the methods disclosed herein or other methods known to those of skill in the art.
EXAMPLES The claimed invention is further illustrated by the following Examples, which should not be construed as limiting. Those of skill in the art will recognize that the claimed invention may be practiced with variations of the disclosed structures, materials, compositions, and methods, and such variations are regarded as within the scope of the claimed invention.
The RTases described herein were overexpressed in E. coli, purified to homogeneity, and tested for their ability to enhance RNA detection in the context of reverse transcriptase quantitative PCR (RT-qPCR).
Example 1. Preparation of Reverse Transcriptase Mutants by Site Directed Mutagenesis a. Cloning of MMLV RTase Mutants Created from Base Construct (RNase H Minus Construct)
MM4LV RTase mutants were prepared by first introducing three mutations (D524G, E562Q, and D583N) into the amino acid sequence of the wild-type, or naturally occurring, MMLV RTase to prepare an MMLV RTase base construct (SEQ TD NO: 637). The three mutations, which are contained in the SuperScript II RTase (Invitrogen), have been shown to reduce RNase H activity (see U.S. Pat. No. 5,405,776). The MMLV RTase base construct was optimized for E. coli expression and obtained as gBlocks® Gene Fragments (Integrated DNA Technologies) or by custom gene synthesis with the appropriate purification tag. Subsequent genes were amplified using standard PCR conditions and primers (see Tables 1 and 21). Amplified DNA was subjected to purification using a QIAquick PCR Purification kit (Qiagen, Catalog #28104), followed by gene fragment assembly into a pET28b expression plasmid. Plasmid DNA was isolated and sequenced to verify the desired sequence following transformation into E. coli cells. MMLV RTase mutations were selected by rational design (FIGS. 1A-1C) and introduced by site-directed mutagenesis, using standard PCR conditions and primers (see Tables 1 and 21). Resulting plasmids were transformed into E. coli BL21(DE3) cells for expression.
TABLE 1
Sequences of primers used for cloning of
MMLV RTase base constructs and mutants
into pET28b.
SEQ
ID Primer Sequence
NO: Primer Name (5′-3′)
1 pET28b 5′ GGTATATCTCCTTCT
Reverse TAAAGTTAAACAAAA
TTATTTCTAGAGGGG
AAT
2 pET28b 3′ GATCCGGCTGCTAAC
Forward AAAGCC
3 MMLV 5′ Primer TTTTGTTTAACTTTA
AGAAGGAGATATACC
ATGGGCAGCAGCCAT
CATCATC
4 MMLV 3′ Primer GCAGCCAACTCAGCT
TCCTTTCGGGCTTTG
TTAAAAATGCTCGCT
AGTGTAGGGAGAGC
5 MMLV K53A Top AAGCACCGTTGATCA
SDM TCCCGTTAGCGGCAA
CGTCTACACCTGTCT
CTATCAAAC
6 MMLV K53R Top AAGCACCGTTGATCA
SDM TCCCGTTACGTGCAA
CGTCTACACCTGTCT
CTATCAAAC
7 MMLV K53E Top AAGCACCGTTGATCA
SDM TCCCGTTAGAAGCAA
CGTCTACACCTGTCT
CTATCAAAC
8 MMLV T55A Top CCGTTGATCATCCCG
SDM TTAAAGGCAGCGTCT
ACACCTGTCTCTATC
AAACAGTACCCC
9 MMLV T55R Top CCGTTGATCATCCCG
SDM TTAAAGGCACGTTCT
ACACCTGTCTCTATC
AAACAGTACCCC
10 MMLV T55E Top CCGTTGATCATCCCG
SDM TTAAAGGCAGAATCT
ACACCTGTCTCTATC
AAACAGTACCCC
11 MMLV T57A Top ATCATCCCGTTAAAG
SDM GCAACGTCTGCGCCT
GTCTCTATCAAACAG
TACCCCATGAG
12 MMLV T57R Top ATCATCCCGTTAAAG
SDM GCAACGTCTCGTCCT
GTCTCTATCAAACAG
TACCCCATGAG
13 MMLV T57E Top ATCATCCCGTTAAAG
SDM GCAACGTCTGAACCT
GTCTCTATCAAACAG
TACCCCATGAG
14 MMLV V59A Top CCGTTAAAGGCAACG
SDM TCTACACCTGCGTCT
ATCAAACAGTACCCC
ATGAGTCAAGAGG
15 MMLV V59R Top CCGTTAAAGGCAACG
SDM TCTACACCTCGTTCT
ATCAAACAGTACCCC
ATGAGTCAAGAGG
16 MMLV V59E Top CCGTTAAAGGCAACG
SDM TCTACACCTGAATCT
ATCAAACAGTACCCC
ATGAGTCAAGAGG
17 MMLV 161A Top TAAAGGCAACGTCTA
SDM CACCTGTCTCTGCGA
AACAGTACCCCATGA
GTCAAGAGG
18 MMLV I61R Top TAAAGGCAACGTCTA
SDM CACCTGTCTCTCGTA
AACAGTACCCCATGA
GTCAAGAGG
19 MMLV 16IE Top TAAAGGCAACGTCTA
SDM CACCTGTCTCTGAAA
AACAGTACCCCATGA
GTCAAGAGG
20 MMLV K62A Top GGCAACGTCTACACC
SDM TGTCTCTATCGCGCA
GTACCCCATGAGTCA
AGAGGC
21 MMLV K62R Top GGCAACGTCTACACC
SDM TGTCTCTATCCGTCA
GTACCCCATGAGTCA
AGAGGC
22 MMLV K62E Top GGCAACGTCTACACC
SDM TGTCTCTATCGAACA
GTACCCCATGAGTCA
AGAGGC
23 MMLV Q68A Top CTGTCTCTATCAAAC
SDM AGTACCCCATGAGTG
CGGAGGCCCGCCTGG
G
24 MMLV Q68R Top CTGTCTCTATCAAAC
SDM AGTACCCCATGAGTC
GTGAGGCCCGCCTGG
G
25 MMLV Q68E Top CTGTCTCTATCAAAC
SDM AGTACCCCATGAGTG
AAGAGGCCCGCCTGG
G
26 MMLV K75A Top GGCCCGCCTGGGGAT
SDM TGCGCCACATATTCA
GCGCTTGCTGGACCA
27 MMLV K75R Top GGCCCGCCTGGGGAT
SDM TCGTCCACATATTCA
GCGCTTGCTGGACCA
28 MMLV K75E Top GGCCCGCCTGGGGAT
SDM TGAACCACATATTCA
GCGCTTGCTGGACCA
29 MMLV Q79A Top CGCCTGGGGATTAAG
SDM CCACATATTGCGCGC
TTGCTGGACCAGGGG
30 MMLV Q79R Top CGCCTGGGGATTAAG
SDM CCACATATTCGTCGC
TTGCTGGACCAGGGG
31 MMLV Q79E Top CGCCTGGGGATTAAG
SDM CCACATATTGAACGC
TTGCTGGACCAGGGG
32 MMLV L99A Top CCGTGGAACACCCCC
SDM CTTGCGCCCGTGAAA
AAGCCAGGTACAAAC
33 MMLV L99R Top CCGTGGAACACCCCC
SDM CTTCGTCCCGTGAAA
AAGCCAGGTACAAAC
34 MMLV L99E Top CCGTGGAACACCCCC
SDM CTTGAACCCGTGAAA
AAGCCAGGTACAAAC
35 MMLV V101A Top CACCCCCCTTCTGCC
SDM CGCGAAAAAGCCAGG
TACAAACGATTATCG
TCC
36 MMLV V101R Top CACCCCCCTTCTGCC
SDM CCGTAAAAAGCCAGG
TACAAACGATTATCG
TCC
37 MMLV V101E Top CACCCCCCTTCTGCC
SDM CGAAAAAAAGCCAGG
TACAAACGATTATCG
TCC
38 MMLV K102A Top CCCCCTTCTGCCCGT
SDM GGCGAAGCCAGGTAC
AAACGATTATCGTCC
39 MMLV K102R Top CCCCCTTCTGCCCGT
SDM GCGTAAGCCAGGTAC
AAACGATTATCGTCC
40 MMLV K102E Top CCCCCTTCTGCCCGT
SDM GGAAAAGCCAGGTAC
AAACGATTATCGTCC
41 MMLV K103A Top CCCCCTTCTGCCCGT
SDM GAAAGCGCCAGGTAC
AAACGATTATCGTCC
AGTT
42 MMLV K103R Top CCCCCTTCTGCCCGT
SDM GAAACGTCCAGGTAC
AAACGATTATCGTCC
AGTT
43 MMLV K103E Top CCCCCTTCTGCCCGT
SDM GAAAGAACCAGGTAC
AAACGATTATCGTCC
AGTT
44 MMLV T106A Top GCCCGTGAAAAAGCC
SDM AGGTGCGAACGATTA
TCGTCCAGTTCAAGA
TCTTCG
45 MMLV T106R Top GCCCGTGAAAAAGCC
SDM AGGTCGTAACGATTA
TCGTCCAGTTCAAGA
TCTTCG
46 MMLV T106E Top GCCCGTGAAAAAGCC
SDM AGGTGAAAACGATTA
TCGTCCAGTTCAAGA
TCTTCG
47 MMLV N107A Top CCCGTGAAAAAGCCA
SDM GGTACAGCGGATTAT
CGTCCAGTTCAAGAT
CTTCGCG
48 MMLV N107R Top CCCGTGAAAAAGCCA
SDM GGTACACGTGATTAT
CGTCCAGTTCAAGAT
CTTCGCG
49 MMLV N107E CCCGTGAAAAAGCCAGGTAC
Top SDM AGAAGATTATCGTCCAGTTC
AAGATCTTCGCG
50 MMLV Y109A CGTGAAAAAGCCAGGTACAA
Top SDM ACGATGCGCGTCCAGTTCAA
GATCTTCGCG
51 MMLV Y109R CGTGAAAAAGCCAGGTACAA
Top SDM ACGATCGTCGTCCAGTTCAA
GATCTTCGCG
52 MMLV Y109E CGTGAAAAAGCCAGGTACAA
Top SDM ACGATGAACGTCCAGTTCAA
GATCTTCGCG
53 MMLVR110A CGTGAAAAAGCCAGGTACAA
Top SDM ACGATTATGCGCCAGTTCAA
GATCTTCGCGAGG
54 MMLVR110K CGTGAAAAAGCCAGGTACAA
Top SDM ACGATTATAAACCAGTTCAA
GATCTTCGCGAGG
55 MMLV R110E CGTGAAAAAGCCAGGTACAA
Top SDM ACGATTATGAACCAGTTCAA
GATCTTCGCGAGG
56 MMLV V112A GCCAGGTACAAACGATTATC
Top SDM GTCCAGCGCAAGATCTTCGC
GAGGTCAACAAAC
57 MMLV VI12R GCCAGGTACAAACGATTATC
Top SDM GTCCACGTCAAGATCTTCGC
GAGGTCAACAAAC
58 MMLV V112E GCCAGGTACAAACGATTATC
Top SDM GTCCAGAACAAGATCTTCGC
GAGGTCAACAAAC
59 MMLV K120A AGTTCAAGATCTTCGCGAGG
Top SDM TCAACGCGCGCGTAGAAGAC
ATCCATCCGAC
60 MMLV K120R AGTTCAAGATCTTCGCGAGG
Top SDM TCAACCGTCGCGTAGAAGAC
ATCCATCCGAC
61 MMLV K120E AGTTCAAGATCTTCGCGAGG
Top SDM TCAACGAACGCGTAGAAGAC
ATCCATCCGAC
62 MMLV El23A GCGAGGTCAACAAACGCGTA
Top SDM GCGGACATCCATCCGACTGT
ACCTAATCC
63 MMLV E123R GCGAGGTCAACAAACGCGTA
Top SDM CGTGACATCCATCCGACTGT
ACCTAATCC
64 MMLV E123D GCGAGGTCAACAAACGCGTA
Top SDM GATGACATCCATCCGACTGT
ACCTAATCC
65 MMLV T128V ACGCGTAGAAGACATCCATC
Top SDM CGGTGGTACCTAATCCTTAT
AATCTGTTATCAGGCCTGC
66 MMLV T128R ACGCGTAGAAGACATCCATC
Top SDM CGCGTGTACCTAATCCTTAT
AATCTGTTATCAGGCCTGC
67 MMLV T128E ACGCGTAGAAGACATCCATC
Top SDM CGGAAGTACCTAATCCTTAT
AATCTGTTATCAGGCCTGC
68 MMLV K193A CGTCTGCCCCAGGGCTTTGC
Top SDM GAACAGCCCCACATTGTTCG
ATGAA
69 MMLV K193R CGTCTGCCCCAGGGCTTTCG
Top SDM TAACAGCCCCACATTGTTCG
ATGAA
70 MMLV K193E CGTCTGCCCCAGGGCTTTGA
Top SDM AAACAGCCCCACATTGTTCG
ATGAA
71 MMLV E282A AGAAGGTCAACGTTGGCTGA
Top SDM CTGCGGCGCGTAAGGAGACC
GTAATG
72 MMLV E282R AGAAGGTCAACGTTGGCTGA
Top SDM CTCGTGCGCGTAAGGAGACC
GTAATG
73 MMLV E282D AGAAGGTCAACGTTGGCTGA
Top SDM CTGATGCGCGTAAGGAGACC
GTAATG
74 MMLV A283V GAAGGTCAACGTTGGCTGAC
Top SDM TGAAGTGCGTAAGGAGACCG
TAATGGGGC
75 MMLV A283R GAAGGTCAACGTTGGCTGAC
Top SDM TGAACGTCGTAAGGAGACCG
TAATGGGGC
76 MMLV A283E GAAGGTCAACGTTGGCTGAC
Top SDM TGAAGAACGTAAGGAGACCG
TAATGGGGC
77 MMLV Q291A GCGTAAGGAGACCGTAATGG
Top SDM GGGCGCCTACGCCTAAGACG
CCACG
78 MMLV Q291R GCGTAAGGAGACCGTAATGG
Top SDM GGCGTCCTACGCCTAAGACG
CCACG
79 MMLV Q291E GCGTAAGGAGACCGTAATGG
Top SDM GGGAACCTACGCCTAAGACG
CCACG
80 MMLV GAGACCGTAATGGGGCAGCC
T293A TGCGCCTAAGACGCCACGCC
Top SDM AGTTG
81 MMLV GAGACCGTAATGGGGCAGCC
T293R TCGTCCTAAGACGCCACGCC
Top SDM AGTTG
82 MMLV GAGACCGTAATGGGGCAGCC
T293E TGAACCTAAGACGCCACGCC
Top SDM AGTTG
83 MMLV K295A GTAATGGGGCAGCCTACGCC
Top SDM TGCGACGCCACGCCAGTTGC
GTGAA
84 MMLV K295R GTAATGGGGCAGCCTACGCC
Top SDM TCGTACGCCACGCCAGTTGC
GTGAA
85 MMLV K295E GTAATGGGGCAGCCTACGCC
Top SDM TGAAACGCCACGCCAGTTGC
GTGAA
86 MMLV TGGGGCAGCCTACGCCTAAG
T296A GCGCCACGCCAGTTGCGTGA
Top SDM ATTTT
87 MMLV TGGGGCAGCCTACGCCTAAG
T296R CGTCCACGCCAGTTGCGTGA
Top SDM ATTTT
88 MMLV TGGGGCAGCCTACGCCTAAG
T296E GAACCACGCCAGTTGCGTGA
Top SDM ATTTT
89 MMLV R298A GCCTACGCCTAAGACGCCAG
Top SDM CGCAGTTGCGTGAATTTTTG
GGCACAG
90 MMLV R298K GCCTACGCCTAAGACGCCAA
Top SDM AACAGTTGCGTGAATTTTTG
GGCACAG
91 MMLV R298E GCCTACGCCTAAGACGCCAG
Top SDM AACAGTTGCGTGAATTTTTG
GGCACAG
92 MMLV R30IA CCTAAGACGCCACGCCAGTT
Top SDM GGCGGAATTTTTGGGCACAG
CGGGA
93 MMLV R301K CCTAAGACGCCACGCCAGTT
Top SDM GAAAGAATTTTTGGGCACAG
CGGGA
94 MMLV R301E CCTAAGACGCCACGCCAGTT
Top SDM GGAAGAATTTTTGGGCACAG
CGGGA
95 MMLV K329A GCACCCCTGTACCCCTTAAC
Top SDM AGCGACAGGGACGCTTTTCA
ACTGG
96 MMLV K329R GCACCCCTGTACCCCTTAAC
Top SDM ACGTACAGGGACGCTTTTCA
ACTGG
97 MMLV K329E GCACCCCTGTACCCCTTAAC
Top SDM AGAAACAGGGACGCTTTTCA
ACTGG
98 MMLV K53A GTTTGATAGAGACAGGTGTA
Btm SDM GACGTTGCCGCTAACGGGAT
GATCAACGGTGCTT
99 MMLV K53R GTTTGATAGAGACAGGTGTA
Btm SDM GACGTTGCACGTAACGGGAT
GATCAACGGTGCTT
100 MMLV K53E GTTTGATAGAGACAGGTGTA
Btm SDM GACGTTGCTTCTAACGGGAT
GATCAACGGTGCTT
101 MMLV GGGGTACTGTTTGATAGAGA
T55A CAGGTGTAGACGCTGCCTTT
Btm SDM AACGGGATGATCAACGG
102 MMLV GGGGTACTGTTTGATAGAGA
T55R CAGGTGTAGAACGTGCCTTT
Btm SDM AACGGGATGATCAACGG
103 MMLV GGGGTACTGTTTGATAGAGA
T55E CAGGTGTAGATTCTGCCTTT
Btm SDM AACGGGATGATCAACGG
104 MMLV CTCATGGGGTACTGTTTGAT
T57A AGAGACAGGCGCAGACGTTG
Btm SDM CCTTTAACGGGATGAT
105 MMLV CTCATGGGGTACTGTTTGAT
T57R AGAGACAGGACGAGACGTTG
Btm SDM CCTTTAACGGGATGAT
106 MMLV CTCATGGGGTACTGTTTGAT
T57E AGAGACAGGTTCAGACGTTG
Btm SDM CCTTTAACGGGATGAT
107 MMLV V59A CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGATAGACGCAGGTGT
AGACGTTGCCTTTAACGG
108 MMLV V59R CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGATAGAACGAGGTGT
AGACGTTGCCTTTAACGG
109 MMLV V59E CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGATAGATTCAGGTGT
AGACGTTGCCTTTAACGG
110 MMLV 161A CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTCGCAGAGACAGGTGT
AGACGTTGCCTTTA
111 MMLV 161R CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTACGAGAGACAGGTGT
AGACGTTGCCTTTA
112 MMLV 16IE CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTTTCAGAGACAGGTGT
AGACGTTGCCTTTA
113 MMLV K62A GCCTCTTGACTCATGGGGTA
Btm SDM CTGCGCGATAGAGACAGGTG
TAGACGTTGCC
114 MMLV K62R GCCTCTTGACTCATGGGGTA
Btm SDM CTGACGGATAGAGACAGGTG
TAGACGTTGCC
115 MMLV K62E GCCTCTTGACTCATGGGGTA
Btm SDM CTGTTCGATAGAGACAGGTG
TAGACGTTGCC
116 MMLV Q68A CTGTCTCTATCAAACAGTAC
Btm SDM CCCATGAGTGCGGAGGCCCG
CCTGGG
117 MMLV Q68R CTGTCTCTATCAAACAGTAC
Btm SDM CCCATGAGTCGTGAGGCCCG
CCTGGG
118 MMLV Q68E CTGTCTCTATCAAACAGTAC
Btm SDM CCCATGAGTGAAGAGGCCCG
CCTGGG
119 MMLV K75A TGGTCCAGCAAGCGCTGAAT
Btm SDM ATGTGGCGCAATCCCCAGGC
GGGCC
120 MMLV K75R TGGTCCAGCAAGCGCTGAAT
Btm SDM ATGTGGACGAATCCCCAGGC
GGGCC
121 MMLV K75E TGGTCCAGCAAGCGCTGAAT
Btm SDM ATGTGGTTCAATCCCCAGGC
GGGCC
122 MMLV Q79A CCCCTGGTCCAGCAAGCGCG
Btm SDM CAATATGTGGCTTAATCCCC
AGGCG
123 MMLV Q79R CCCCTGGTCCAGCAAGCGAC
Btm SDM GAATATGTGGCTTAATCCCC
AGGCG
124 MMLV Q79E CCCCTGGTCCAGCAAGCGTT
Btm SDM CAATATGTGGCTTAATCCCC
AGGCG
125 MMLV L99A GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGCGCAAGGGGGGTGTTC
CACGG
126 MMLV L99R GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGACGAAGGGGGGTGTTC
CACGG
127 MMLV L99E GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGTTCAAGGGGGGTGTTC
CACGG
128 MMLV V101A GGACGATAATCGTTTGTACC
Btm SDM TGGCTTTTTCGCGGGCAGAA
GGGGGGTG
129 MMLV VI01R GGACGATAATCGTTTGTACC
Btm SDM TGGCTTTTTACGGGGCAGAA
GGGGGGTG
130 MMLV V101E GGACGATAATCGTTTGTACC
Btm SDM TGGCTTTTTTTCGGGCAGAA
GGGGGGTG
131 MMLV K102A GGACGATAATCGTTTGTACC
Btm SDM TGGCTTCGCCACGGGCAGAA
GGGGG
132 MMLV K102R GGACGATAATCGTTTGTACC
Btm SDM TGGCTTACGCACGGGCAGAA
GGGGG
133 MMLV K102E GGACGATAATCGTTTGTACC
Btm SDM TGGCTTTTCCACGGGCAGAA
GGGGG
134 MMLV K103 A AACTGGACGATAATCGTTTG
Btm SDM TACCTGGCGCTTTCACGGGC
AGAAGGGGG
135 MMLV K103R AACTGGACGATAATCGTTTG
Btm SDM TACCTGGACGTTTCACGGGC
AGAAGGGGG
136 MMLV K103E AACTGGACGATAATCGTTTG
Btm SDM TACCTGGTTCTTTCACGGGC
AGAAGGGGG
137 MMLV CGAAGATCTTGAACTGGACG
T106A ATAATCGTTCGCACCTGGCT
Btm SDM TTTTCACGGGC
138 MMLV CGAAGATCTTGAACTGGACG
T106R ATAATCGTTACGACCTGGCT
Btm SDM TTTTCACGGGC
139 MMLV CGAAGATCTTGAACTGGACG
T106E ATAATCGTTTTCACCTGGCT
Btm SDM TTTTCACGGGC
140 MMLVN107A CGCGAAGATCTTGAACTGGA
Btm SDM CGATAATCCGCTGTACCTGG
CTTTTTCACGGG
141 MMLV N107R CGCGAAGATCTTGAACTGGA
Btm SDM CGATAATCACGTGTACCTGG
CTTTTTCACGGG
142 MMLV N107E CGCGAAGATCTTGAACTGGA
Btm SDM CGATAATCTTCTGTACCTGG
CTTTTTCACGGG
143 MMLV Y109A CGCGAAGATCTTGAACTGGA
Btm SDM CGCGCATCGTTTGTACCTGG
CTTTTTCACG
144 MMLV Y109R CGCGAAGATCTTGAACTGGA
Btm SDM CGACGATCGTTTGTACCTGG
CTTTTTCACG
145 MMLV Y109E CGCGAAGATCTTGAACTGGA
Btm SDM CGTTCATCGTTTGTACCTGG
CTTTTTCACG
146 MMLVR110A CCTCGCGAAGATCTTGAACT
Btm SDM GGCGCATAATCGTTTGTACC
TGGCTTTTTCACG
147 MMLV R110K CCTCGCGAAGATCTTGAACT
Btm SDM GGTTTATAATCGTTTGTACC
TGGCTTTTTCACG
148 MMLVR110E CCTCGCGAAGATCTTGAACT
Btm SDM GGTTCATAATCGTTTGTACC
TGGCTTTTTCACG
149 MMLV V112A GTTTGTTGACCTCGCGAAGA
Btm SDM TCTTGCGCTGGACGATAATC
GTTTGTACCTGGC
150 MMLV V112R GTTTGTTGACCTCGCGAAGA
Btm SDM TCTTGACGTGGACGATAATC
GTTTGTACCTGGC
151 MMLV VI12E GTTTGTTGACCTCGCGAAGA
Btm SDM TCTTGTTCTGGACGATAATC
GTTTGTACCTGGC
152 MMLV K120A GTCGGATGGATGTCTTCTAC
Btm SDM GCGCGCGTTGACCTCGCGAA
GATCTTGAACT
153 MMLV K120R GTCGGATGGATGTCTTCTAC
Btm SDM GCGACGGTTGACCTCGCGAA
GATCTTGAACT
154 MMLV K120E GTCGGATGGATGTCTTCTAC
Btm SDM GCGTTCGTTGACCTCGCGAA
GATCTTGAACT
155 MMLV E123A GGATTAGGTACAGTCGGATG
Btm SDM GATGTCCGCTACGCGTTTGT
TGACCTCGC
156 MMLV E123R GGATTAGGTACAGTCGGATG
Btm SDM GATGTCACGTACGCGTTTGT
TGACCTCGC
157 MMLV E123D GGATTAGGTACAGTCGGATG
Btm SDM GATGTCATCTACGCGTTTGT
TGACCTCGC
158 MMLV GCAGGCCTGATAACAGATTA
T128V TAAGGATTAGGTACCACCGG
Btm SDM ATGGATGTCTTCTACGCGT
159 MMLV GCAGGCCTGATAACAGATTA
T128R TAAGGATTAGGTACACGCGG
Btm SDM ATGGATGTCTTCTACGCGT
160 MMLV GCAGGCCTGATAACAGATTA
T128E TAAGGATTAGGTACTTCCGG
Btm SDM ATGGATGTCTTCTACGCGT
161 MMLV K193A TTCATCGAACAATGTGGGGC
Btm SDM TGTTCGCAAAGCCCTGGGGC
AGACG
162 MMLV K193R TTCATCGAACAATGTGGGGC
Btm SDM TGTTACGAAAGCCCTGGGGC
AGACG
163 MMLV K193E TTCATCGAACAATGTGGGGC
Btm SDM TGTTTTCAAAGCCCTGGGGC
AGACG
164 MMLV E282A CATTACGGTCTCCTTACGCG
Btm SDM CCGCAGTCAGCCAACGTTGA
CCTTCT
165 MMLV E282R CATTACGGTCTCCTTACGCG
Btm SDM CACGAGTCAGCCAACGTTGA
CCTTCT
166 MMLV E282D CATTACGGTCTCCTTACGCG
Btm SDM CATCAGTCAGCCAACGTTGA
CCTTCT
167 MMLV A283V GCCCCATTACGGTCTCCTTA
Btm SDM CGCACTTCAGTCAGCCAACG
TTGACCTTC
168 MMLV A283R GCCCCATTACGGTCTCCTTA
Btm SDM CGACGTTCAGTCAGCCAACG
TTGACCTTC
169 MMLV A283E GCCCCATTACGGTCTCCTTA
Btm SDM CGTTCTTCAGTCAGCCAACG
TTGACCTTC
170 MMLV Q29IA CGTGGCGTCTTAGGCGTAGG
Btm SDM CGCCCCCATTACGGTCTCCT
TACGC
171 MMLV Q291R CGTGGCGTCTTAGGCGTAGG
Btm SDM ACGCCCCATTACGGTCTCCT
TACGC
172 MMLV Q291E CGTGGCGTCTTAGGCGTAGG
Btm SDM TTCCCCCATTACGGTCTCCT
TACGC
173 MMLV T293A CAACTGGCGTGGCGTCTTAG
Btm SDM GCGCAGGCTGCCCCATTACG
GTCTC
174 MMLV T293R CAACTGGCGTGGCGTCTTAG
Btm SDM GACGAGGCTGCCCCATTACG
GTCTC
175 MMLV T293E CAACTGGCGTGGCGTCTTAG
Btm SDM GTTCAGGCTGCCCCATTACG
GTCTC
176 MMLV K295A TTCACGCAACTGGCGTGGCG
Btm SDM TCGCAGGCGTAGGCTGCCCC
ATTAC
177 MMLV K295R TTCACGCAACTGGCGTGGCG
Btm SDM TACGAGGCGTAGGCTGCCCC
ATTAC
178 MMLV K295E TTCACGCAACTGGCGTGGCG
Btm SDM TTTCAGGCGTAGGCTGCCCC
ATTAC
179 MMLV T296A AAAATTCACGCAACTGGCGT
Btm SDM GGCGCCTTAGGCGTAGGCTG
CCCCA
180 MMLV T296R AAAATTCACGCAACTGGCGT
Btm SDM GGACGCTTAGGCGTAGGCTG
CCCCA
181 MMLV T296E AAAATTCACGCAACTGGCGT
Btm SDM GGTTCCTTAGGCGTAGGCTG
CCCCA
182 MMLV R298A CTGTGCCCAAAAATTCACGC
Btm SDM AACTGCGCTGGCGTCTTAGG
CGTAGGC
183 MMLV R298K CTGTGCCCAAAAATTCACGC
Btm SDM AACTGTTTTGGCGTCTTAGG
CGTAGGC
184 MMLV R298E CTGTGCCCAAAAATTCACGC
Btm SDM AACTGTTCTGGCGTCTTAGG
CGTAGGC
185 MMLV R301A TCCCGCTGTGCCCAAAAATT
Btm SDM CCGCCAACTGGCGTGGCGTC
TTAGG
186 MMLV R301K TCCCGCTGTGCCCAAAAATT
Btm SDM CTTTCAACTGGCGTGGCGTC
TTAGG
187 MMLV R301E TCCCGCTGTGCCCAAAAATT
Btm SDM CTTCCAACTGGCGTGGCGTC
TTAGG
188 MMLV K329A CCAGTTGAAAAGCGTCCCTG
Btm SDM TCGCTGTTAAGGGGTACAGG
GGTGC
189 MMLV K329R CCAGTTGAAAAGCGTCCCTG
Btm SDM TACGTGTTAAGGGGTACAGG
GGTGC
190 MMLV K329E CCAGTTGAAAAGCGTCCCTG
Btm SDM TTTCTGTTAAGGGGTACAGG
GGTGC
191 MMLV I61G TAAAGGCAACGTCTACACCT
Top SDM GTCTCTGGCAAACAGTACCC
CATGAGTCAAGAGG
192 MMLV 161G CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGCCAGAGACAGGTGT
AGACGTTGCCTTTA
193 MMLV 161L TAAAGGCAACGTCTACACCT
Top SDM GTCTCTCTGAAACAGTACCC
CATGAGTCAAGAGG
194 MMLV 161L CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTCAGAGAGACAGGTGT
AGACGTTGCCTTTA
195 MMLV 16IV TAAAGGCAACGTCTACACCT
Top SDM GTCTCTGTGAAACAGTACCC
CATGAGTCAAGAGG
196 MMLV 16IV CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTCACAGAGACAGGTGT
AGACGTTGCCTTTA
197 MMLV 16IP TAAAGGCAACGTCTACACCT
Top SDM GTCTCTCCGAAACAGTACCC
CATGAGTCAAGAGG
198 MMLV 16IP CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTCGGAGAGACAGGTGT
AGACGTTGCCTTTA
199 MMLV 161M TAAAGGCAACGTCTACACCT
Top SDM GTCTCTATGAAACAGTACCC
CATGAGTCAAGAGG
200 MMLV 161M CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTCATAGAGACAGGTGT
AGACGTTGCCTTTA
201 MMLV 16IS TAAAGGCAACGTCTACACCT
Top SDM GTCTCTAGCAAACAGTACCC
CATGAGTCAAGAGG
202 MMLV 16IS CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGCTAGAGACAGGTGT
AGACGTTGCCTTTA
203 MMLV 16IT TAAAGGCAACGTCTACACCT
Top SDM GTCTCTACCAAACAGTACCC
CATGAGTCAAGAGG
204 MMLV 16IT CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGGTAGAGACAGGTGT
AGACGTTGCCTTTA
205 MMLV 161C TAAAGGCAACGTCTACACCT
Top SDM GTCTCTTGCAAACAGTACCC
CATGAGTCAAGAGG
206 MMLV 161C CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGCAAGAGACAGGTGT
AGACGTTGCCTTTA
207 MMLV 16IF TAAAGGCAACGTCTACACCT
Top SDM GTCTCTTTTAAACAGTACCC
CATGAGTCAAGAGG
208 MMLV 161F CCTCTTGACTCATGGGGTAC
Btm TGTTTAAAAGAGACA
SDM GGTGTAGACGTTGCCTTTA
209 MMLV 161Y TAAAGGCAACGTCTACACCT
Top SDM GTCTCTTATAAACAGTACCC
CATGAGTCAAGAGG
210 MMLV 161Y CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTATAAGAGACAGGTGT
AGACGTTGCCTTTA
211 MMLV 161H TAAAGGCAACGTCTACACCT
Top SDM GTCTCTCATAAACAGTACCC
CATGAGTCAAGAGG
212 MMLV 161H CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTATGAGAGACAGGTGT
AGACGTTGCCTTTA
213 MMLV I61W TAAAGGCAACGTCTACACCT
Top SDM GTCTCTTGGAAACAGTACCC
CATGAGTCAAGAGG
214 MMLV 161W CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTCCAAGAGACAGGTGT
AGACGTTGCCTTTA
215 MMLV 161D TAAAGGCAACGTCTACACCT
Top SDM GTCTCTGATAAACAGTACCC
CATGAGTCAAGAGG
216 MMLV 16ID CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTATCAGAGACAGGTGT
AGACGTTGCCTTTA
217 MMLV 16IN TAAAGGCAACGTCTACACCT
Top SDM GTCTCTAACAAACAGTACCC
CATGAGTCAAGAGG
218 MMLV 16IN CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTGTTAGAGACAGGTGT
AGACGTTGCCTTTA
219 MMLV 16IQ TAAAGGCAACGTCTACACCT
Top SDM GTCTCTCAGAAACAGTACCC
CATGAGTCAAGAGG
220 MMLV 16IQ CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTCTGAGAGACAGGTGT
AGACGTTGCCTTTA
221 MMLV 161K TAAAGGCAACGTCTACACCT
Top SDM GTCTCTAAAAAACAGTACCC
CATGAGTCAAGAGG
222 MMLV 16IK CCTCTTGACTCATGGGGTAC
Btm SDM TGTTTTTTAGAGACAGGTGT
AGACGTTGCCTTTA
223 MMLV Q68G CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTGGCGAGGCCCG
CCTGGG
224 MMLV Q68G CCCAGGCGGGCCTCGCCACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
225 MMLV Q68L CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTCTGGAGGCCCG
CCTGGG
226 MMLV Q68L CCCAGGCGGGCCTCCAGACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
227 MMLV Q68I CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTATTGAGGCCCG
CCTGGG
228 MMLV Q68I CCCAGGCGGGCCTCAATACT
Btm CATGGGGTACTGTTT
SDM GATAGAGACAG
229 MMLV Q68V CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTGTGGAGGCCCG
CCTGGG
230 MMLV Q68V CCCAGGCGGGCCTCCACACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
231 MMLV Q68P CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTCCGGAGGCCCG
CCTGGG
232 MMLV Q68P CCCAGGCGGGCCTCCGGACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
233 MMLV Q68M CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTATGGAGGCCCG
CCTGGG
234 MMLV Q68M CCCAGGCGGGCCTCCATACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
235 MMLV Q68S CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTAGCGAGGCCCG
CCTGGG
236 MMLV Q68S CCCAGGCGGGCCTCGCTACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
237 MMLV Q68T CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTACCGAGGCCCG
CCTGGG
238 MMLV Q68T CCCAGGCGGGCCTCGGTACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
239 MMLV Q68C CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTTGCGAGGCCCG
CCTGGG
240 MMLV Q68C CCCAGGCGGGCCTCGCAACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
241 MMLV Q68F CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTTTTGAGGCCCG
CCTGGG
242 MMLV Q68F CCCAGGCGGGCCTCAAAACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
243 MMLV Q68Y CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTTATGAGGCCCG
CCTGGG
244 MMLV Q68Y CCCAGGCGGGCCTCATAACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
245 MMLV Q68H CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTCATGAGGCCCG
CCTGGG
246 MMLV Q68H CCCAGGCGGGCCTCATGACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
247 MMLV Q68W CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTTGGGAGGCCCG
CCTGGG
248 MMLV Q68W CCCAGGCGGGCCTCCCAACT
Btm CATGGGGTACTGTTT
SDM GATAGAGACAG
249 MMLV Q68D CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTGATGAGGCCCG
CCTGGG
250 MMLV Q68D CCCAGGCGGGCCTCATCACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
251 MMLV Q68N CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTAACGAGGCCCG
CCTGGG
252 MMLV Q68N CCCAGGCGGGCCTCGTTACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
253 MMLV Q68K CTGTCTCTATCAAACAGTAC
Top SDM CCCATGAGTAAAGAGGCCCG
CCTGGG
254 MMLV Q68K CCCAGGCGGGCCTCTTTACT
Btm SDM CATGGGGTACTGTTTGATAG
AGACAG
255 MMLV Q79G CGCCTGGGGATTAAGCCACA
Top SDM TATTGGCCGCTTGCTGGACC
AGGGG
256 MMLV Q79G CCCCTGGTCCAGCAAGCGGC
Btm SDM CAATATGTGGCTTAATCCCC
AGGCG
257 MMLV Q79L CGCCTGGGGATTAAGCCACA
Top SDM TATTCTGCGCTTGCTGGACC
AGGGG
258 MMLV Q79L CCCCTGGTCCAGCAAGCGCA
Btm SDM GAATATGTGGCTTAATCCCC
AGGCG
259 MMLV Q79I CGCCTGGGGATTAAGCCACA
Top SDM TATTATTCGCTTGCTGGACC
AGGGG
260 MMLV Q79I CCCCTGGTCCAGCAAGCGAA
Btm SDM TAATATGTGGCTTAATCCCC
AGGCG
261 MMLV Q79V CGCCTGGGGATTAAGCCACA
Top SDM TATTGTGCGCTTGCTGGACC
AGGGG
262 MMLV Q79V CCCCTGGTCCAGCAAGCGCA
Btm SDM CAATATGTGGCTTAATCCCC
AGGCG
263 MMLV Q79P CGCCTGGGGATTAAGCCACA
Top SDM TATTCCGCGCTTGCTGGACC
AGGGG
264 MMLV Q79P CCCCTGGTCCAGCAAGCGCG
Btm SDM GAATATGTGGCTTAATCCCC
AGGCG
265 MMLV Q79M CGCCTGGGGATTAAGCCACA
Top SDM TATTATGCGCTTGCTGGACC
AGGGG
266 MMLV Q79M CCCCTGGTCCAGCAAGCGCA
Btm SDM TAATATGTGGCTTAATCCCC
AGGCG
267 MMLV Q79S CGCCTGGGGATTAAGCCACA
Top SDM TATTAGCCGCTTGCTGGACC
AGGGG
268 MMLV Q79S CCCCTGGTCCAGCAAGCGGC
Btm TAATATGTGGCTTAA
SDM TCCCCAGGCG
269 MMLV Q79T CGCCTGGGGATTAAGCCACA
Top SDM TATTACCCGCTTGCTGGACC
AGGGG
270 MMLV Q79T CCCCTGGTCCAGCAAGCGGG
Btm SDM TAATATGTGGCTTAATCCCC
AGGCG
271 MMLV Q79C CGCCTGGGGATTAAGCCACA
Top SDM TATTTGCCGCTTGCTGGACC
AGGGG
272 MMLV Q79C CCCCTGGTCCAGCAAGCGGC
Btm SDM AAATATGTGGCTTAATCCCC
AGGCG
273 MMLV Q79F CGCCTGGGGATTAAGCCACA
Top SDM TATTTTTCGCTTGCTGGACC
AGGGG
274 MMLV Q79F CCCCTGGTCCAGCAAGCGAA
Btm SDM AAATATGTGGCTTAATCCCC
AGGCG
275 MMLV Q79Y CGCCTGGGGATTAAGCCACA
Top SDM TATTTATCGCTTGCTGGACC
AGGGG
276 MMLV Q79Y CCCCTGGTCCAGCAAGCGAT
Btm SDM AAATATGTGGCTTAATCCCC
AGGCG
277 MMLV Q79H CGCCTGGGGATTAAGCCACA
Top SDM TATTCATCGCTTGCTGGACC
AGGGG
278 MMLV Q79H CCCCTGGTCCAGCAAGCGAT
Btm SDM GAATATGTGGCTTAATCCCC
AGGCG
279 MMLV Q79W CGCCTGGGGATTAAGCCACA
Top SDM TATTTGGCGCTTGCTGGACC
AGGGG
280 MMLV Q79W CCCCTGGTCCAGCAAGCGCC
Btm SDM AAATATGTGGCTTAATCCCC
AGGCG
281 MMLV Q79D CGCCTGGGGATTAAGCCACA
Top SDM TATTGATCGCTTGCTGGACC
AGGGG
282 MMLV Q79D CCCCTGGTCCAGCAAGCGAT
Btm SDM CAATATGTGGCTTAATCCCC
AGGCG
283 MMLV Q79N CGCCTGGGGATTAAGCCACA
Top SDM TATTAACCGCTTGCTGGACC
AGGGG
284 MMLV Q79N CCCCTGGTCCAGCAAGCGGT
Btm SDM TAATATGTGGCTTAATCCCC
AGGCG
285 MMLV Q79K CGCCTGGGGATTAAGCCACA
Top SDM TATTAAACGCTTGCTGGACC
AGGGG
286 MMLV Q79K CCCCTGGTCCAGCAAGCGTT
Btm SDM TAATATGTGGCTTAATCCCC
AGGCG
287 MMLV L99G CCGTGGAACACCCCCCTTGG
Top SDM CCCCGTGAAAAAGCCAGGTA
CAAAC
288 MMLV L99G GTTTGTACCTGGCTTTTTCA
Btm CGGGGCCAAGGGGGG
SDM TGTTCCACGG
289 MMLV L99I CCGTGGAACACCCCCCTTAT
Top SDM TCCCGTGAAAAAGCCAGGTA
CAAAC
290 MMLV L99I GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGAATAAGGGGGGTGTTC
CACGG
291 MMLV L99V CCGTGGAACACCCCCCTTGT
Top SDM GCCCGTGAAAAAGCCAGGTA
CAAAC
292 MMLV L99V GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGCACAAGGGGGGTGTTC
CACGG
293 MMLV L99P CCGTGGAACACCCCCCTTCC
Top SDM GCCCGTGAAAAAGCCAGGTA
CAAAC
294 MMLV L99P GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGCGGAAGGGGGGTGTTC
CACGG
295 MMLV L99M CCGTGGAACACCCCCCTTAT
Top SDM GCCCGTGAAAAAGCCAGGTA
CAAAC
296 MMLV L99M GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGCATAAGGGGGGTGTTC
CACGG
297 MMLV L99S CCGTGGAACACCCCCCTTAG
Top SDM CCCCGTGAAAAAGCCAGGTA
CAAAC
298 MMLV L99S GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGGCTAAGGGGGGTGTTC
CACGG
299 MMLV L99T CCGTGGAACACCCCCCTTAC
Top SDM CCCCGTGAAAAAGCCAGGTA
CAAAC
300 MMLV L99T GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGGGTAAGGGGGGTGTTC
CACGG
301 MMLV L99C CCGTGGAACACCCCCCTTTG
Top SDM CCCCGTGAAAAAGCCAGGTA
CAAAC
302 MMLV L99C GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGGCAAAGGGGGGTGTTC
CACGG
303 MMLV L99F CCGTGGAACACCCCCCTTTT
Top SDM TCCCGTGAAAAAGCCAGGTA
CAAAC
304 MMLV L99F GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGAAAAAGGGGGGTGTTC
CACGG
305 MMLV L99Y CCGTGGAACACCCCCCTTTA
Top SDM TCCCGTGAAAAAGCCAGGTA
CAAAC
306 MMLV L99Y GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGATAAAGGGGGGTGTTC
CACGG
307 MMLV L99H CCGTGGAACACCCCCCTTCA
Top SDM TCCCGTGAAAAAGCCAGGTA
CAAAC
308 MMLV L99H GTTTGTACCTGGCTTTTTCA
Btm CGGGATGAAGGGGGG
SDM TGTTCCACGG
309 MMLV L99W CCGTGGAACACCCCCCTTTG
Top SDM GCCCGTGAAAAAGCCAGGTA
CAAAC
310 MMLV L99W GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGCCAAAGGGGGGTGTTC
CACGG
311 MMLV L99D CCGTGGAACACCCCCCTTGA
Top SDM TCCCGTGAAAAAGCCAGGTA
CAAAC
312 MMLV L99D GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGATCAAGGGGGGTGTTC
CACGG
313 MMLV L99N CCGTGGAACACCCCCCTTAA
Top SDM CCCCGTGAAAAAGCCAGGTA
CAAAC
314 MMLV L99N GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGGTTAAGGGGGGTGTTC
CACGG
315 MMLV L99Q CCGTGGAACACCCCCCTTCA
Top SDM GCCCGTGAAAAAGCCAGGTA
CAAAC
316 MMLV L99Q GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGCTGAAGGGGGGTGTTC
CACGG
317 MMLV L99K CCGTGGAACACCCCCCTTAA
Top SDM ACCCGTGAAAAAGCCAGGTA
CAAAC
318 MMLV L99K GTTTGTACCTGGCTTTTTCA
Btm SDM CGGGTTTAAGGGGGGTGTTC
CACGG
319 MMLV E282G AGAAGGTCAACGTTGGCTGA
Top SDM CTGGCGCGCGTAAGGAGACC
GTAATG
320 MMLV E282G CATTACGGTCTCCTTACGCG
Btm SDM CGCCAGTCAGCCAACGTTGA
CCTTCT
321 MMLV E282L AGAAGGTCAACGTTGGCTGA
Top SDM CTCTGGCGCGTAAGGAGACC
GTAATG
322 MMLV E282L CATTACGGTCTCCTTACGCG
Btm SDM CCAGAGTCAGCCAACGTTGA
CCTTCT
323 MMLV E282I AGAAGGTCAACGTTGGCTGA
Top SDM CTATTGCGCGTAAGGAGACC
GTAATG
324 MMLV E282I CATTACGGTCTCCTTACGCG
Btm SDM CAATAGTCAGCCAACGTTGA
CCTTCT
325 MMLV E282V AGAAGGTCAACGTTGGCTGA
Top SDM CTGTGGCGCGTAAGGAGACC
GTAATG
326 MMLV E282V CATTACGGTCTCCTTACGCG
Btm SDM CCACAGTCAGCCAACGTTGA
CCTTCT
327 MMLV E282R AGAAGGTCAACGTTGGCTGA
Top SDM CTCCGGCGCGTAAGGAGACC
GTAATG
328 MMLV E282P CATTACGGTCTCCTTACGCG
Btm CCGGAGTCAGCCAAC
SDM GTTGACCTTCT
329 MMLV E282M AGAAGGTCAACGTTGGCTGA
Top SDM CTATGGCGCGTAAGGAGACC
GTAATG
330 MMLV E282M CATTACGGTCTCCTTACGCG
Btm SDM CCATAGTCAGCCAACGTTGA
CCTTCT
331 MMLV E282S AGAAGGTCAACGTTGGCTGA
Top SDM CTAGCGCGCGTAAGGAGACC
GTAATG
332 MMLV E282S CATTACGGTCTCCTTACGCG
Btm SDM CGCTAGTCAGCCAACGTTGA
CCTTCT
333 MMLV E282T AGAAGGTCAACGTTGGCTGA
Top SDM CTACCGCGCGTAAGGAGACC
GTAATG
334 MMLV E282T CATTACGGTCTCCTTACGCG
Btm SDM CGGTAGTCAGCCAACGTTGA
CCTTCT
335 MMLV E282C AGAAGGTCAACGTTGGCTGA
Top SDM CTTGCGCGCGTAAGGAGACC
GTAATG
336 MMLV E282C CATTACGGTCTCCTTACGCG
Btm SDM CGCAAGTCAGCCAACGTTGA
CCTTCT
337 MMLV E282F AGAAGGTCAACGTTGGCTGA
Top SDM CTTTTGCGCGTAAGGAGACC
GTAATG
338 MMLV E282F CATTACGGTCTCCTTACGCG
Btm SDM CAAAAGTCAGCCAACGTTGA
CCTTCT
339 MMLV E282Y AGAAGGTCAACGTTGGCTGA
Top SDM CTTATGCGCGTAAGGAGACC
GTAATG
340 MMLV E282Y CATTACGGTCTCCTTACGCG
Btm SDM CATAAGTCAGCCAACGTTGA
CCTTCT
341 MMLV E282H AGAAGGTCAACGTTGGCTGA
Top SDM CTCATGCGCGTAAGGAGACC
GTAATG
342 MMLV E282H CATTACGGTCTCCTTACGCG
Btm SDM CATGAGTCAGCCAACGTTGA
CCTTCT
343 MMLV E282W AGAAGGTCAACGTTGGCTGA
Top SDM CTTGGGCGCGTAAGGAGACC
GTAATG
344 MMLV E282W CATTACGGTCTCCTTACGCG
Btm SDM CCCAAGTCAGCCAACGTTGA
CCTTCT
345 MMLV E282N AGAAGGTCAACGTTGGCTGA
Top SDM CTAACGCGCGTAAGGAGACC
GTAATG
346 MMLV E282N CATTACGGTCTCCTTACGCG
Btm SDM CGTTAGTCAGCCAACGTTGA
CCTTCT
347 MMLV E282Q AGAAGGTCAACGTTGGCTGA
Top SDM CTCAGGCGCGTAAGGAGACC
GTAATG
348 MMLV E282Q CATTACGGTCTCCTTACGCG
Btm CCTGAGTCAGCCAAC
SDM GTTGACCTTCT
349 MMLV E282K AGAAGGTCAACGTTGGCTGA
Top SDM CTAAAGCGCGTAAGGAGACC
GTAATG
350 MMLV E282K CATTACGGTCTCCTTACGCG
Btm SDM CTTTAGTCAGCCAACGTTGA
CCTTCT
351 MMLV R298G GCCTACGCCTAAGACGCCAG
Top SDM GCCAGTTGCGTGAATTTTTG
GGCACAG
352 MMLV R298G CTGTGCCCAAAAATTCACGC
Btm SDM AACTGGCCTGGCGTCTTAGG
CGTAGGC
353 MMLV R298L GCCTACGCCTAAGACGCCAC
Top SDM TGCAGTTGCGTGAATTTTTG
GGCACAG
354 MMLV R298L CTGTGCCCAAAAATTCACGC
Btm SDM AACTGCAGTGGCGTCTTAGG
CGTAGGC
355 MMLV R298I GCCTACGCCTAAGACGCCAA
Top SDM TTCAGTTGCGTGAATTTTTG
GGCACAG
356 MMLV R298I CTGTGCCCAAAAATTCACGC
Btm SDM AACTGAATTGGCGTCTTAGG
CGTAGGC
357 MMLV R298V GCCTACGCCTAAGACGCCAG
Top SDM TGCAGTTGCGTGAATTTTTG
GGCACAG
358 MMLV R298V CTGTGCCCAAAAATTCACGC
Btm SDM AACTGCACTGGCGTCTTAGG
CGTAGGC
359 MMLV R298P GCCTACGCCTAAGACGCCAC
Top SDM CGCAGTTGCGTGAATTTTTG
GGCACAG
360 MMLV R298P CTGTGCCCAAAAATTCACGC
Btm SDM AACTGCGGTGGCGTCTTAGG
CGTAGGC
361 MMLV R298M GCCTACGCCTAAGACGCCAA
Top SDM TGCAGTTGCGTGAATTTTTG
GGCACAG
362 MMLV R298M CTGTGCCCAAAAATTCACGC
Btm SDM AACTGCATTGGCGTCTTAGG
CGTAGGC
363 MMLV R298S GCCTACGCCTAAGACGCCAA
Top SDM GCCAGTTGCGTGAATTTTTG
GGCACAG
364 MMLV R298S CTGTGCCCAAAAATTCACGC
Btm SDM AACTGGCTTGGCGTCTTAGG
CGTAGGC
365 MMLV R298T GCCTACGCCTAAGACGCCAA
Top SDM CCCAGTTGCGTGAATTTTTG
GGCACAG
366 MMLV R298T CTGTGCCCAAAAATTCACGC
Btm SDM AACTGGGTTGGCGTCTTAGG
CGTAGGC
367 MMLV R298C GCCTACGCCTAAGACGCCAT
Top SDM GCCAGTTGCGTGAATTTTTG
GGCACAG
368 MMLV R298C CTGTGCCCAAAAATTCACGC
Btm AACTGGCATGGCGTC
SDM TTAGGCGTAGGC
369 MMLV R298F GCCTACGCCTAAGACGCCAT
Top SDM TTCAGTTGCGTGAATTTTTG
GGCACAG
370 MMLV R298F CTGTGCCCAAAAATTCACGC
Btm SDM AACTGAAATGGCGTCTTAGG
CGTAGGC
371 MMLV R298Y GCCTACGCCTAAGACGCCAT
Top SDM ATCAGTTGCGTGAATTTTTG
GGCACAG
372 MMLV R298Y CTGTGCCCAAAAATTCACGC
Btm SDM AACTGATATGGCGTCTTAGG
CGTAGGC
373 MMLV R298H GCCTACGCCTAAGACGCCAC
Top SDM ATCAGTTGCGTGAATTTTTG
GGCACAG
374 MMLV R298H CTGTGCCCAAAAATTCACGC
Btm SDM AACTGATGTGGCGTCTTAGG
CGTAGGC
375 MMLV R298W GCCTACGCCTAAGACGCCAT
Top SDM GGCAGTTGCGTGAATTTTTG
GGCACAG
376 MMLV R298W CTGTGCCCAAAAATTCACGC
Btm SDM AACTGCCATGGCGTCTTAGG
CGTAGGC
377 MMLV R298D GCCTACGCCTAAGACGCCAG
Top SDM ATCAGTTGCGTGAATTTTTG
GGCACAG
378 MMLV R298D CTGTGCCCAAAAATTCACGC
Btm SDM AACTGATCTGGCGTCTTAGG
CGTAGGC
379 MMLV R298N GCCTACGCCTAAGACGCCAA
Top SDM ACCAGTTGCGTGAATTTTTG
GGCACAG
380 MMLV R298N CTGTGCCCAAAAATTCACGC
Btm SDM AACTGGTTTGGCGTCTTAGG
CGTAGGC
381 MMLV R298Q GCCTACGCCTAAGACGCCAC
Top SDM AGCAGTTGCGTGAATTTTTG
GGCACAG
382 MMLV R298Q CTGTGCCCAAAAATTCACGC
Btm SDM AACTGCTGTGGCGTCTTAGG
CGTAGGC
383 MMLV I61R/Q68R AGGCAACGTCTACACCTGTC
Top SDM TCTCGTAAACAGTACCCCAT
GAGTCGTGAGGCCCGCCTGG
GG
384 MMLV I61R/Q68R CCCCAGGCGGGCCTCACGAC
Btm SDM TCATGGGGTACTGTTTACGA
GAGACAGGTGTAGACGTTGC
CT
385 MMLV I61K/Q68R AGGCAACGTCTACACCTGTC
Top SDM TCTAAAAAACAGTACCCCAT
GAGTCGTGAGG
386 MMLV I61K/Q68R CCTCACGACTCATGGGGTAC
Btm SDM TGTTTTTTAGAGACAGGTGT
AGACGTTGCCT
387 MMLV I61M/Q68R AGGCAACGTCTACACCTGTC
Top SDM TCTATGAAACAGTACCCCAT
GAGTCGTGAGG
388 MMLV I61M/Q68R CCTCACGACTCATGGGGTAC
TGTTTCATAGAGACA
Btm SDM GGTGTAGACGTTGCCT
389 MMLV I61M/Q68I AGGCAACGTCTACACCTGTC
Top SDM TCTATGAAACAGTACCCCAT
GAGTATTGAGGCC
390 MMLV I61M/Q68I GGCCTCAATACTCATGGGGT
Btm SDM ACTGTTTCATAGAGACAGGT
GTAGACGTTGCCT
393 MMLV 5′ Primer GTCTCTATCAAACAGTACCC
CATGGCGCAAGAGGCCCGCC
TGGG
394 MMLV 3’ Primer GTCTCTATCAAACAGTACCC
CATGCGTCAAGAGGCCCGCC
TGGG
395 MMLV G73A CATGAGTCAAGAGGCCCGCG
Top SDM AGGGGATTAAGCCACATATT
CAGCG
396 MMLV G73R GAGTCAAGAGGCCCGCCTGG
Top SDM CGATTAAGCCACATATTCAG
CGCTTGC
397 MMLV G73E GAGTCAAGAGGCCCGCCTGC
Top SDM GTATTAAGCCACATATTCAG
CGCTTGC
398 MMLV P76A GAGTCAAGAGGCCCGCCTGG
Top SDM AGATTAAGCCACATATTCAG
CGCTTGC
399 MMLV P76R GGCCCGCCTGGGGATTAAGG
Top SDM CGCATATTCAGCGCTTGCTG
GACC
400 MMLV P76E GGCCCGCCTGGGGATTAAGC
Top SDM GTCATATTCAGCGCTTGCTG
GACC
401 MMLV H77A GGCCCGCCTGGGGATTAAGG
Top SDM AGCATATTCAGCGCTTGCTG
GACC
402 MMLV H77R CCGCCTGGGGATTAAGCCAG
Top SDM CGATTCAGCGCTTGCTGGAC
CAG
403 MMLV H77E CCGCCTGGGGATTAAGCCAC
Top SDM GTATTCAGCGCTTGCTGGAC
CAG
404 MMLV L82A CCGCCTGGGGATTAAGCCAG
Top SDM AGATTCAGCGCTTGCTGGAC
CAG
405 MMLV L82R GATTAAGCCACATATTCAGC
Top SDM GCTTGGCGGACCAGGGGATC
TTGGTCC
406 MMLV L82E GATTAAGCCACATATTCAGC
Top SDM GCTTGCGTGACCAGGGGATC
TTGGTCC
407 MMLV D83A GATTAAGCCACATATTCAGC
Top SDM GCTTGGAGGACCAGGGGATC
TTGGTCC
408 MMLV D83R GCCACATATTCAGCGCTTGC
Top SDM TGGCGCAGGGGATCTTGGTC
CCATG
409 MMLV D83E GCCACATATTCAGCGCTTGC
Top SDM TGCGTCAGGGGATCTTGGTC
CCATG
410 MMLV I125A GCCACATATTCAGCGCTTGC
Top TGGAGCAGGGGATCT
SDM TGGTCCCATG
411 MMLV I125R AGGTCAACAAACGCGTAGAA
Top SDM GACGCGCATCCGACTGTACC
TAATCCTTATAAT
412 MMLV I125E AGGTCAACAAACGCGTAGAA
Top SDM GACCGTCATCCGACTGTACC
TAATCCTTATAAT
413 MMLV V129A AGGTCAACAAACGCGTAGAA
Top SDM GACGAGCATCCGACTGTACC
TAATCCTTATAAT
414 MMLV V129R GCGTAGAAGACATCCATCCG
Top SDM ACTGCGCCTAATCCTTATAA
TCTGTTATCAGGC
415 MMLV V129E GCGTAGAAGACATCCATCCG
Top SDM ACTCGTCCTAATCCTTATAA
TCTGTTATCAGGC
416 MMLV LI 98A GCGTAGAAGACATCCATCCG
Top SDM ACTGAGCCTAATCCTTATAA
TCTGTTATCAGGC
417 MMLV LI98R AGGGCTTTAAAAACAGCCCC
Top SDM ACAGCGTTCGATGAAGCACT
TCACCGTGA
418 MMLV L198E AGGGCTTTAAAAACAGCCCC
Top SDM ACACGTTTCGATGAAGCACT
TCACCGTGA
419 MMLV E201A AGGGCTTTAAAAACAGCCCC
Top SDM ACAGAGTTCGATGAAGCACT
TCACCGTGA
420 MMLV E201R TTTAAAAACAGCCCCACATT
Top SDM GTTCGATGCGGCACTTCACC
GTGACTTAGCAG
421 MMLV E201D TTTAAAAACAGCCCCACATT
Top SDM GTTCGATCGTGCACTTCACC
GTGACTTAGCAG
422 MMLV R205A TTTAAAAACAGCCCCACATT
Top SDM GTTCGATGATGCACTTCACC
GTGACTTAGCAG
423 MMLV R205K CACATTGTTCGATGAAGCAC
Top SDM TTCACGCGGACTTAGCAGAC
TTCCGTATCCA
424 MMLV R205E CACATTGTTCGATGAAGCAC
Top SDM TTCACAAAGACTTAGCAGAC
TTCCGTATCCA
425 MMLV D209A GATGAAGCACTTCACCGTGA
Top SDM CTTAGAGGACTTCCGTATCC
AACACCCAG
426 MMLV D209R AAGCACTTCACCGTGACTTA
Top SDM GCAGCGTTCCGTATCCAACA
CCCAGACTT
427 MMLV D209E AAGCACTTCACCGTGACTTA
Top SDM GCACGTTTCCGTATCCAACA
CCCAGACTT
428 MMLV F210A AAGCACTTCACCGTGACTTA
Top SDM GCAGAGTTCCGTATCCAACA
CCCAGACTT
429 MMLV F2I0R CACTTCACCGTGACTTAGCA
Top SDM GACGCGCGTATCCAACACCC
AGACTTAATTC
430 MMLV F210E CACTTCACCGTGACTTAGCA
Top GACCGTCGTATCCAA
SDM CACCCAGACTTAATTC
431 MMLV R211A CACTTCACCGTGACTTAGCA
Top SDM GACGAGCGTATCCAACACCC
AGACTTAATTC
432 MMLV R211K TTCACCGTGACTTAGCAGAC
Top SDM TTCGCGATCCAACACCCAGA
CTTAATTCTGTTA
433 MMLV R21 IE TTCACCGTGACTTAGCAGAC
Top SDM TTCAAAATCCAACACCCAGA
CTTAATTCTGTTA
434 MMLV 1212A TTCACCGTGACTTAGCAGAC
Top SDM TTCGAGATCCAACACCCAGA
CTTAATTCTGTTA
435 MMLV I212R CCGTGACTTAGCAGACTTCC
Top SDM GTGCGCAACACCCAGACTTA
ATTCTGTTACAG
436 MMLV I212E CCGTGACTTAGCAGACTTCC
Top SDM GTCGTCAACACCCAGACTTA
ATTCTGTTACAG
437 MMLV Q213A CCGTGACTTAGCAGACTTCC
Top SDM GTGAGCAACACCCAGACTTA
ATTCTGTTACAG
438 MMLV Q213R GTGACTTAGCAGACTTCCGT
Top SDM ATCGCGCACCCAGACTTAAT
TCTGTTACAGTAT
439 MMLV Q213E GTGACTTAGCAGACTTCCGT
Top SDM ATCCGTCACCCAGACTTAAT
TCTGTTACAGTAT
440 MMLV K348A GTGACTTAGCAGACTTCCGT
Top SDM ATCGAGCACCCAGACTTAAT
TCTGTTACAGTAT
441 MMLV K348R AGCAAAAGGCGTATCAGGAG
Top SDM ATCGCGCAAGCTTTGTTGAC
CGCACCC
442 MMLV K348E AGCAAAAGGCGTATCAGGAG
Top SDM ATCCGTCAAGCTTTGTTGAC
CGCACCC
443 MMLV L352A AGCAAAAGGCGTATCAGGAG
Top SDM ATCGAGCAAGCTTTGTTGAC
CGCACCC
444 MMLV L352R CGTATCAGGAGATCAAACAA
Top SDM GCTTTGGCGACCGCACCCGC
GTTGGG
445 MMLV L352E CGTATCAGGAGATCAAACAA
Top SDM GCTTTGCGTACCGCACCCGC
GTTGGG
446 MMLV K285A CGTATCAGGAGATCAAACAA
Top SDM GCTTTGGAGACCGCACCCGC
GTTGGG
447 MMLV K285R GTTGGCTGACTGAAGCGCGT
Top SDM GCGGAGACCGTAATGGGGCA
GC
448 MMLV K285E GTTGGCTGACTGAAGCGCGT
Top SDM CGTGAGACCGTAATGGGGCA
GC
449 MMLV Q299A GTTGGCTGACTGAAGCGCGT
Top SDM GAGGAGACCGTAATGGGGCA
GC
450 MMLV Q299R TACGCCTAAGACGCCACGCG
CGTTGCGTGAATTTT
Top SDM TGGGCACAGC
451 MMLV Q299E TACGCCTAAGACGCCACGCC
Top SDM GTTTGCGTGAATTTTTGGGC
ACAGC
452 MMLV G308A TACGCCTAAGACGCCACGCG
Top SDM AGTTGCGTGAATTTTTGGGC
ACAGC
453 MMLV G308R GCGTGAATTTTTGGGCACAG
Top SDM CGGCGTTCTGTCGTTTATGG
ATTCCTGGG
454 MMLV G308E GCGTGAATTTTTGGGCACAG
Top SDM CGCGTTTCTGTCGTTTATGG
ATTCCTGGG
455 MMLV R311A GCGTGAATTTTTGGGCACAG
Top SDM CGGAGTTCTGTCGTTTATGG
ATTCCTGGG
456 MMLV R311K GGGCACAGCGGGATTCTGTG
Top SDM CGTTATGGATTCCTGGGTTC
GCTGA
457 MMLV R311E GGGCACAGCGGGATTCTGTA
Top SDM AATTATGGATTCCTGGGTTC
GCTGA
458 MMLV Y271A GGGCACAGCGGGATTCTGTG
Top SDM AGTTATGGATTCCTGGGTTC
GCTGA
459 MMLV Y271R GTCAAAAACAGGTAAAGTAC
Top SDM CTTGGGGCGTTGCTGAAAGA
AGGTCAACGTTGG
460 MMLV Y271E GTCAAAAACAGGTAAAGTAC
Top SDM CTTGGGCGTTTGCTGAAAGA
AGGTCAACGTTGG
461 MMLV L280A GTCAAAAACAGGTAAAGTAC
Top SDM CTTGGGGAGTTGCTGAAAGA
AGGTCAACGTTGG
462 MMLV L280R TGCTGAAAGAAGGTCAACGT
Top SDM TGGGCGACTGAAGCGCGTAA
GGAGACC
463 MMLV L280E TGCTGAAAGAAGGTCAACGT
Top SDM TGGCGTACTGAAGCGCGTAA
GGAGACC
464 MMLV L357A TGCTGAAAGAAGGTCAACGT
Top SDM TGGGAGACTGAAGCGCGTAA
GGAGACC
465 MMLV L357R TTTGTTGACCGCACCCGCGG
Top SDM CGGGTCTTCCGGATTTAACC
AAGCC
466 MMLV L357E TTTGTTGACCGCACCCGCGC
Top SDM GTGGTCTTCCGGATTTAACC
AAGCC
467 MMLV T328A TTTGTTGACCGCACCCGCGG
Top SDM AGGGTCTTCCGGATTTAACC
AAGCC
468 MMLV T328R CTGCACCCCTGTACCCCTTA
Top SDM GCGAAAACAGGGACGCTTTT
CAACTGG
469 MMLV T328E CTGCACCCCTGTACCCCTTA
Top SDM CGTAAAACAGGGACGCTTTT
CAACTGG
470 MMLV G331A CTGCACCCCTGTACCCCTTA
GAGAAAACAGGGACG
Top SDM CTTTTCAACTGG
471 MMLV G331R CCCCTGTACCCCTTAACAAA
Top SDM AACAGCGACGCTTTTCAACT
GGGGGCC
472 MMLV G331E CCCCTGTACCCCTTAACAAA
Top SDM AACACGTACGCTTTTCAACT
GGGGGCC
473 MMLV T332A CCCCTGTACCCCTTAACT'L
Top SDM AAAACAGAGACGCTTTTCAA
CTGGGGGCC
474 MMLV T332R CTGTACCCCTTAACAAAAAC
Top SDM AGGGGCGCTTTTCAACTGGG
GGCCAGAC
475 MMLV T332E CTGTACCCCTTAACAAAAAC
Top SDM AGGGCGTCTTTTCAACTGGG
GGCCAGAC
476 MMLV N335A CTGTACCCCTTAACAAAAAC
Top SDM AGGGGAGCTTTTCAACTGGG
GGCCAGAC
All MMLV N335R CCTTAACAAAAACAGGGACG
Top SDM CTTTTCGCGTGGGGGCCAGA
CCAGCAAA
478 MMLV N335E CCTTAACAAAAACAGGGACG
Top SDM CTTTTCCGTTGGGGGCCAGA
CCAGCAAA
479 MMLV E367A CTTCCGGATTTAACCAAGCC
Top SDM CTTTGCGCTGTTCGTTGATG
AAAAACAGGGATAT
480 MMLV E367R CTTCCGGATTTAACCAAGCC
Top SDM CTTTCGTCTGTTCGTTGATG
AAAAACAGGGATAT
481 MMLV E367D CTTCCGGATTTAACCAAGCC
Top SDM CTTTGATCTGTTCGTTGATG
AAAAACAGGGATAT
482 MMLV F369A GATTTAACCAAGCCCTTTGA
Top SDM GCTGGCGGTTGATGAAAAAC
AGGGATATGCAAAAG
483 MMLV F369R GATTTAACCAAGCCCTTTGA
Top SDM GCTGCGTGTTGATGAAAAAC
AGGGATATGCAAAAG
484 MMLV F369E GATTTAACCAAGCCCTTTGA
Top SDM GCTGGAGGTTGATGAAAAAC
AGGGATATGCAAAAG
485 MMLV R389A CCCAAAAGTTAGGCCCGTGG
Top SDM GCGCGCCCTGTTGCTTACTT
GAGTAA
486 MMLV R389K CCCAAAAGTTAGGCCCGTGG
Top SDM AAACGCCCTGTTGCTTACTT
GAGTAA
487 MMLV R389E CCCAAAAGTTAGGCCCGTGG
Top SDM GAGCGCCCTGTTGCTTACTT
GAGTAA
488 MMLV V433A AGTTGACGATGGGTCAACCC
Top SDM TTAGCGATCTTGGCTCCACA
TGCTGTAGA
489 MMLV V433R AGTTGACGATGGGTCAACCC
Top SDM TTACGTATCTTGGCTCCACA
TGCTGTAGA
490 MMLV V433E AGTTGACGATGGGTCAACCC
Top TTAGAGATCTTGGCT
SDM CCACATGCTGTAGA
491 MMLV V476A GGATCGTGTACAATTTGGAC
Top SDM CAGTTGCGGCTTTGAATCCA
GCTACTTTGCTTC
492 MMLV V476R GGATCGTGTACAATTTGGAC
Top SDM CAGTTCGTGCTTTGAATCCA
GCTACTTTGCTTC
493 MMLV V476E GGATCGTGTACAATTTGGAC
Top SDM CAGTTGAGGCTTTGAATCCA
GCTACTTTGCTTC
494 MMLV 1593A CGTTATGCTTTTGCAACAGC
Top SDM GCATGCGCATGGCGAAATTT
ACCGCCGC
495 MMLV 1593R CGTTATGCTTTTGCAACAGC
Top SDM GCATCGTCATGGCGAAATTT
ACCGCCGC
496 MMLV I593E CGTTATGCTTTTGCAACAGC
Top SDM GCATGAGCATGGCGAAATTT
ACCGCCGC
497 MMLV E596A GCAACAGCGCATATCCATGG
Top SDM CGCGATTTACCGCCGCCGTG
GTC
498 MMLV E596R GCAACAGCGCATATCCATGG
Top SDM CCGTATTTACCGCCGCCGTG
GTC
499 MMLV E596D GCAACAGCGCATATCCATGG
Top SDM CGATATTTACCGCCGCCGTG
GTC
500 MMLV 1597A CAACAGCGCATATCCATGGC
Top SDM GAAGCGTACCGCCGCCGTGG
TCTG
501 MMLV 1597R CAACAGCGCATATCCATGGC
Top SDM GAACGTTACCGCCGCCGTGG
TCTG
502 MMLV I597E CAACAGCGCATATCCATGGC
Top SDM GAAGAGTACCGCCGCCGTGG
TCTG
503 MMLV R650A AGCGGAGGCTCGTGGAAACG
Top SDM CGATGGCGGACCAAGCTGCC
C
504 MMLV R650K AGCGGAGGCTCGTGGAAACA
Top SDM AAATGGCGGACCAAGCTGCC
C
505 MMLV R650E AGCGGAGGCTCGTGGAAACG
Top SDM AGATGGCGGACCAAGCTGCC
C
506 MMLV Q654A GTGGAAACCGTATGGCGGAC
Top SDM GCGGCTGCCCGTAAGGCGGC
507 MMLV Q654R GTGGAAACCGTATGGCGGAC
Top SDM CGTGCTGCCCGTAAGGCGGC
508 MMLV Q654E GTGGAAACCGTATGGCGGAC
Top SDM GAGGCTGCCCGTAAGGCGGC
509 MMLV R657A TATGGCGGACCAAGCTGCCG
Top SDM CGAAGGCGGCGATCACAGAG
AC
510 MMLV R657K TATGGCGGACCAAGCTGCCA
AAAAGGCGGCGATCA
Top SDM CAGAGAC
511 MMLV R657E TATGGCGGACCAAGCTGCCG
Top SDM AGAAGGCGGCGATCACAGAG
AC
512 MMLV G73A GCAAGCGCTGAATATGTGGC
Btm SDM TTAATCGCCAGGCGGGCCTC
TTGACTC
513 MMLV G73R GCAAGCGCTGAATATGTGGC
Btm SDM TTAATACGCAGGCGGGCCTC
TTGACTC
514 MMLV G73E GCAAGCGCTGAATATGTGGC
Btm SDM TTAATCTCCAGGCGGGCCTC
TTGACTC
515 MMLV P76A GGTCCAGCAAGCGCTGAATA
Btm SDM TGCGCCTTAATCCCCAGGCG
GGCC
516 MMLV P76R GGTCCAGCAAGCGCTGAATA
Btm SDM TGACGCTTAATCCCCAGGCG
GGCC
517 MMLV P76E GGTCCAGCAAGCGCTGAATA
Btm SDM TGCTCCTTAATCCCCAGGCG
GGCC
518 MMLV H77A CTGGTCCAGCAAGCGCTGAA
Btm SDM TCGCTGGCTTAATCCCCAGG
CGG
519 MMLV H77R CTGGTCCAGCAAGCGCTGAA
Btm SDM TACGTGGCTTAATCCCCAGG
CGG
520 MMLV H77E CTGGTCCAGCAAGCGCTGAA
Btm SDM TCTCTGGCTTAATCCCCAGG
CGG
521 MMLV L82A GGACCAAGATCCCCTGGTCC
Btm SDM GCCAAGCGCTGAATATGTGG
CTTAATC
522 MMLV L82R GGACCAAGATCCCCTGGTCA
Btm SDM CGCAAGCGCTGAATATGTGG
CTTAATC
523 MMLV L82E GGACCAAGATCCCCTGGTCC
Btm SDM TCCAAGCGCTGAATATGTGG
CTTAATC
524 MMLV D83A CATGGGACCAAGATCCCCTG
Btm SDM CGCCAGCAAGCGCTGAATAT
GTGGC
525 MMLV D83R CATGGGACCAAGATCCCCTG
Btm SDM ACGCAGCAAGCGCTGAATAT
GTGGC
526 MMLV D83E CATGGGACCAAGATCCCCTG
Btm SDM CTCCAGCAAGCGCTGAATAT
GTGGC
527 MMLV I125A ATTATAAGGATTAGGTACAG
Btm SDM TCGGATGCGCGTCTTCTACG
CGTTTGTTGACCT
528 MMLV I125R ATTATAAGGATTAGGTACAG
Btm SDM TCGGATGACGGTCTTCTACG
CGTTTGTTGACCT
529 MMLV I125E ATTATAAGGATTAGGTACAG
Btm SDM TCGGATGCTCGTCTTCTACG
CGTTTGTTGACCT
530 MMLV VI29A GCCTGATAACAGATTATAAG
GATTAGGCGCAGTCG
Btm SDM GATGGATGTCTTCTACGC
531 MMLV V129R GCCTGATAACAGATTATAAG
Btm SDM GATTAGGACGAGTCGGATGG
ATGTCTTCTACGC
532 MMLV V129E GCCTGATAACAGATTATAAG
Btm SDM GATTAGGCTCAGTCGGATGG
ATGTCTTCTACGC
533 MMLV L198A TCACGGTGAAGTGCTTCATC
Btm SDM GAACGCTGTGGGGCTGTTTT
TAAAGCCCT
534 MMLV L198R TCACGGTGAAGTGCTTCATC
Btm SDM GAAACGTGTGGGGCTGTTTT
TAAAGCCCT
535 MMLV L198E TCACGGTGAAGTGCTTCATC
Btm SDM GAACTCTGTGGGGCTGTTTT
TAAAGCCCT
536 MMLV E201A CTGCTAAGTCACGGTGAAGT
Btm SDM GCCGCATCGAACAATGTGGG
GCTGTTTTTAAA
537 MMLV E201R CTGCTAAGTCACGGTGAAGT
Btm SDM GCACGATCGAACAATGTGGG
GCTGTTTTTAAA
538 MMLV E201D CTGCTAAGTCACGGTGAAGT
Btm SDM GCATCATCGAACAATGTGGG
GCTGTTTTTAAA
539 MMLV R205A TGGATACGGAAGTCTGCTAA
Btm SDM GTCCGCGTGAAGTGCTTCAT
CGAACAATGTG
540 MMLV R205K TGGATACGGAAGTCTGCTAA
Btm SDM GTCTTTGTGAAGTGCTTCAT
CGAACAATGTG
541 MMLV R205E TGGATACGGAAGTCTGCTAA
Btm SDM GTCCTCGTGAAGTGCTTCAT
CGAACAATGTG
542 MMLV D209A AAGTCTGGGTGTTGGATACG
Btm SDM GAACGCTGCTAAGTCACGGT
GAAGTGCTT
543 MMLV D209R AAGTCTGGGTGTTGGATACG
Btm SDM GAAACGTGCTAAGTCACGGT
GAAGTGCTT
544 MMLV D209E AAGTCTGGGTGTTGGATACG
Btm SDM GAACTCTGCTAAGTCACGGT
GAAGTGCTT
545 MMLV F210A GAATTAAGTCTGGGTGTTGG
Btm SDM ATACGCGCGTCTGCTAAGTC
ACGGTGAAGTG
546 MMLV F210R GAATTAAGTCTGGGTGTTGG
Btm SDM ATACGACGGTCTGCTAAGTC
ACGGTGAAGTG
547 MMLV F210E GAATTAAGTCTGGGTGTTGG
Btm SDM ATACGCTCGTCTGCTAAGTC
ACGGTGAAGTG
548 MMLV R211A TAACAGAATTAAGTCTGGGT
Btm SDM GTTGGATCGCGAAGTCTGCT
AAGTCACGGTGAA
549 MMLV R211K TAACAGAATTAAGTCTGGGT
Btm SDM GTTGGATTTTGAAGTCTGCT
AAGTCACGGTGAA
550 MMLV R211E TAACAGAATTAAGTCTGGGT
Btm SDM GTTGGATCTCGAAGT
CTGCTAAGTCACGGTGAA
551 MMLV I2I2A CTGTAACAGAATTAAGTCTG
Btm SDM GGTGTTGCGCACGGAAGTCT
GCTAAGTCACGG
552 MMLV 1212R CTGTAACAGAATTAAGTCTG
Btm SDM GGTGTTGACGACGGAAGTCT
GCTAAGTCACGG
553 MMLV 1212E CTGTAACAGAATTAAGTCTG
Btm SDM GGTGTTGCTCACGGAAGTCT
GCTAAGTCACGG
554 MMLV Q213A ATACTGTAACAGAATTAAGT
Btm SDM CTGGGTGCGCGATACGGAAG
TCTGCTAAGTCAC
555 MMLV Q213R ATACTGTAACAGAATTAAGT
Btm SDM CTGGGTGACGGATACGGAAG
TCTGCTAAGTCAC
556 MMLV Q213E ATACTGTAACAGAATTAAGT
Btm SDM CTGGGTGCTCGATACGGAAG
TCTGCTAAGTCAC
557 MMLV K348A GGGTGCGGTCAACAAAGCTT
Btm SDM GCGCGATCTCCTGATACGCC
TTTTGCT
558 MMLV K348R GGGTGCGGTCAACAAAGCTT
Btm SDM GACGGATCTCCTGATACGCC
TTTTGCT
559 MMLV K348E GGGTGCGGTCAACAAAGCTT
Btm SDM GCTCGATCTCCTGATACGCC
TTTTGCT
560 MMLV L352A CCCAACGCGGGTGCGGTCGC
Btm SDM CAAAGCTTGTTTGATCTCCT
GATACG
561 MMLV L352R CCCAACGCGGGTGCGGTACG
Btm SDM CAAAGCTTGTTTGATCTCCT
GATACG
562 MMLV L352E CCCAACGCGGGTGCGGTCTC
Btm SDM CAAAGCTTGTTTGATCTCCT
GATACG
563 MMLV K285A GCTGCCCCATTACGGTCTCC
Btm SDM GCACGCGCTTCAGTCAGCCA
AC
564 MMLV K285R GCTGCCCCATTACGGTCTCA
Btm SDM CGACGCGCTTCAGTCAGCCA
AC
565 MMLV K285E GCTGCCCCATTACGGTCTCC
Btm SDM TCACGCGCTTCAGTCAGCCA
AC
566 MMLV Q299A GCTGTGCCCAAAAATTCACG
Btm SDM CAACGCGCGTGGCGTCTTAG
GCGTA
567 MMLV Q299R GCTGTGCCCAAAAATTCACG
Btm SDM CAAACGGCGTGGCGTCTTAG
GCGTA
568 MMLV Q299E GCTGTGCCCAAAAATTCACG
Btm SDM CAACTCGCGTGGCGTCTTAG
GCGTA
569 MMLV G308A CCCAGGAATCCATAAACGAC
Btm SDM AGAACGCCGCTGTGCCCAAA
AATTCACGC
570 MMLV G308R CCCAGGAATCCATAAACGAC
AGAAACGCGCTGTGC
Btm SDM CCAAAAATTCACGC
571 MMLV G308E CCCAGGAATCCATAAACGAC
Btm SDM AGAACTCCGCTGTGCCCAAA
AATTCACGC
572 MMLV R311A TCAGCGAACCCAGGAATCCA
Btm SDM TAACGCACAGAATCCCGCTG
TGCCC
573 MMLV R311K TCAGCGAACCCAGGAATCCA
Btm SDM TAATTTACAGAATCCCGCTG
TGCCC
574 MMLV R311E TCAGCGAACCCAGGAATCCA
Btm SDM TAACTCACAGAATCCCGCTG
TGCCC
575 MMLV Y271A CCAACGTTGACCTTCTTTCA
Btm SDM GCAACGCCCCAAGGTACTTT
ACCTGTTTTTGAC
576 MMLV Y271R CCAACGTTGACCTTCTTTCA
Btm SDM GCAAACGCCCAAGGTACTTT
ACCTGTTTTTGAC
577 MMLV Y271E CCAACGTTGACCTTCTTTCA
Btm SDM GCAACTCCCCAAGGTACTTT
ACCTGTTTTTGAC
578 MMLV L280A GGTCTCCTTACGCGCTTCAG
Btm SDM TCGCCCAACGTTGACCTTCT
TTCAGCA
579 MMLV L280R GGTCTCCTTACGCGCTTCAG
Btm SDM TACGCCAACGTTGACCTTCT
TTCAGCA
580 MMLV L280E GGTCTCCTTACGCGCTTCAG
Btm SDM TCTCCCAACGTTGACCTTCT
TTCAGCA
581 MMLV L357A GGCTTGGTTAAATCCGGAAG
Btm SDM ACCCGCCGCGGGTGCGGTCA
ACAAA
582 MMLV L357R GGCTTGGTTAAATCCGGAAG
Btm SDM ACCACGCGCGGGTGCGGTCA
ACAAA
583 MMLV L357E GGCTTGGTTAAATCCGGAAG
Btm SDM ACCCTCCGCGGGTGCGGTCA
ACAAA
584 MMLV T328A CCAGTTGAAAAGCGTCCCTG
Btm SDM TTTTCGCTAAGGGGTACAGG
GGTGCAG
585 MMLV T328R CCAGTTGAAAAGCGTCCCTG
Btm SDM TTTTACGTAAGGGGTACAGG
GGTGCAG
586 MMLV T328E CCAGTTGAAAAGCGTCCCTG
Btm SDM TTTTCTCTAAGGGGTACAGG
GGTGCAG
587 MMLV G331A GGCCCCCAGTTGAAAAGCGT
Btm SDM CGCTGTTTTTGTTAAGGGGT
ACAGGGG
588 MMLV G331R GGCCCCCAGTTGAAAAGCGT
Btm SDM ACGTGTTTTTGTTAAGGGGT
ACAGGGG
589 MMLV G331E GGCCCCCAGTTGAAAAGCGT
Btm SDM CTCTGTTTTTGTTAAGGGGT
ACAGGGG
590 MMLV T332A GTCTGGCCCCCAGTTGAAAA
GCGCCCCTGTTTTTG
Btm SDM TTAAGGGGTACAG
591 MMLV T332R GTCTGGCCCCCAGTTGAAAA
Btm SDM GACGCCCTGTTTTTGTTAAG
GGGTACAG
592 MMLV T332E GTCTGGCCCCCAGTTGAAAA
Btm SDM GCTCCCCTGTTTTTGTTAAG
GGGTACAG
593 MMLV N335A TTTGCTGGTCTGGCCCCCAC
Btm SDM GCGAAAAGCGTCCCTGTTTT
TGTTAAGG
594 MMLV N335R TTTGCTGGTCTGGCCCCCAA
Btm SDM CGGAAAAGCGTCCCTGTTTT
TGTTAAGG
595 MMLV N335E TTTGCTGGTCTGGCCCCCAC
Btm SDM TCGAAAAGCGTCCCTGTTTT
TGTTAAGG
596 MMLV E367A ATATCCCTGTTTTTCATCAA
Btm SDM CGAACAGCGCAAAGGGCTTG
GTTAAATCCGGAAG
597 MMLV E367R ATATCCCTGTTTTTCATCAA
Btm SDM CGAACAGACGAAAGGGCTTG
GTTAAATCCGGAAG
598 MMLV E367D ATATCCCTGTTTTTCATCAA
Btm SDM CGAACAGATCAAAGGGCTTG
GTTAAATCCGGAAG
599 MMLV F369A CTTTTGCATATCCCTGTTTT
Btm SDM TCATCAACCGCCAGCTCAAA
GGGCTTGGTTAAATC
600 MMLV F369R CTTTTGCATATCCCTGTTTT
Btm SDM TCATCAACACGCAGCTCAAA
GGGCTTGGTTAAATC
601 MMLV F369E CTTTTGCATATCCCTGTTTT
Btm SDM TCATCAACCTCCAGCTCAAA
GGGCTTGGTTAAATC
602 MMLV R389A TTACTCAAGTAAGCAACAGG
Btm SDM GCGCGCCCACGGGCCTAACT
TTTGGG
603 MMLV R389K TTACTCAAGTAAGCAACAGG
Btm SDM GCGTTTCCACGGGCCTAACT
TTTGGG
604 MMLV R389E TTACTCAAGTAAGCAACAGG
Btm SDM GCGCTCCCACGGGCCTAACT
TTTGGG
605 MMLV V433A TCTACAGCATGTGGAGCCAA
Btm SDM GATCGCTAAGGGTTGACCCA
TCGTCAACT
606 MMLV V433R TCTACAGCATGTGGAGCCAA
Btm SDM GATACGTAAGGGTTGACCCA
TCGTCAACT
607 MMLV V433E TCTACAGCATGTGGAGCCAA
Btm SDM GATCTCTAAGGGTTGACCCA
TCGTCAACT
608 MMLV V476A GAAGCAAAGTAGCTGGATTC
Btm SDM AAAGCCGCAACTGGTCCAAA
TTGTACACGATCC
609 MMLV V476R GAAGCAAAGTAGCTGGATTC
Btm SDM AAAGCACGAACTGGTCCAAA
TTGTACACGATCC
610 MMLV V476E GAAGCAAAGTAGCTGGATTC
Btm SDM AAAGCCTCAACTGGT
CCAAATTGTACACGATCC
611 MMLV 1593A GCGGCGGTAAATTTCGCCAT
Btm SDM GCGCATGCGCTGTTGCAAAA
GCATAACG
612 MMLV I593R GCGGCGGTAAATTTCGCCAT
Btm SDM GACGATGCGCTGTTGCAAAA
GCATAACG
613 MMLV I593E GCGGCGGTAAATTTCGCCAT
Btm SDM GCTCATGCGCTGTTGCAAAA
GCATAACG
614 MMLV E596A GACCACGGCGGCGGTAAATC
Btm SDM GCGCCATGGATATGCGCTGT
TGC
615 MMLV E596R GACCACGGCGGCGGTAAATA
Btm SDM CGGCCATGGATATGCGCTGT
TGC
616 MMLV E596D GACCACGGCGGCGGTAAATA
Btm SDM TCGCCATGGATATGCGCTGT
TGC
617 MMLV 1597A CAGACCACGGCGGCGGTACG
Btm SDM CTTCGCCATGGATATGCGCT
GTTG
618 MMLV I597R CAGACCACGGCGGCGGTAAC
Btm SDM GTTCGCCATGGATATGCGCT
GTTG
619 MMLV I597E CAGACCACGGCGGCGGTACT
Btm SDM CTTCGCCATGGATATGCGCT
GTTG
620 MMLV R650A GGGCAGCTTGGTCCGCCATC
Btm SDM GCGTTTCCACGAGCCTCCGC
T
621 MMLV R650K GGGCAGCTTGGTCCGCCATT
Btm SDM TTGTTTCCACGAGCCTCCGC
T
622 MMLV R650E GGGCAGCTTGGTCCGCCATC
Btm SDM TCGTTTCCACGAGCCTCCGC
T
623 MMLV Q654A GCCGCCTTACGGGCAGCCGC
Btm SDM GTCCGCCATACGGTTTCCAC
624 MMLV Q654R GCCGCCTTACGGGCAGCACG
Btm SDM GTCCGCCATACGGTTTCCAC
625 MMLV Q654E GCCGCCTTACGGGCAGCCTC
Btm SDM GTCCGCCATACGGTTTCCAC
626 MMLV R657A GTCTCTGTGATCGCCGCCTT
Btm SDM CGCGGCAGCTTGGTCCGCCA
TA
627 MMLV R657K GTCTCTGTGATCGCCGCCTT
Btm SDM TTTGGCAGCTTGGTCCGCCA
TA
628 MMLV R657E GTCTCTGTGATCGCCGCCTT
Btm SDM CTCGGCAGCTTGGTCCGCCA
TA
629 MMLV L280R ATTTGCTGAAAGAAGGTCAA
Top SDM V2 CGTTGGCGTACTGATGCGCG
TAAGGAGACC
630 MMLV L280R GGTCTCCTTACGCGCATCAG
Btm SDM V2 TACGCCAACGTTGACCTTCT
TTCAGCAAAT
631 MMLV L82R GGGATTAAGCCACATATTCG
Top SDM V2 TCGCTTGCGTGACCAGGGGA
TCTTGGTCCC
632 MMLV L82R GGGACCAAGATCCCCTGGTC
Btm SDM V2 ACGCAAGCGACGAATATGTG
GCTTAATCCC
Example 2: Preparation of Reverse Transcriptase Mutants for Screening Increased Activity and Thermostability a. Overexpression of MMLV RTase and Mutant Variants
A test induction was used to determine optimum growing conditions. A colony, with the appropriate strain, was used to inoculate Terrific Broth (TB) media (50 mL) with kanamycin (0.05 mg/mL) and grown at 37° C. until an OD of approximately 0.9 was reached. The 50 mL culture was divided in half to accommodate two induction temperatures. IPTG (1M; 12.5 μL) was used to induce protein expression, followed by growth at two induction temperatures for 21 hours. Aliquots (normalized to an OD of 1.25) were taken at 3 and 21 hours, cells were harvested at 13,000×g for one minute, and harvested cells were stored at −20° C. Cells were resuspended in 1× SDS-PAGE running buffer (270 μL) and 5× SDS-PAGE loading dye (70 μL). Samples were boiled for 5 minutes, sonicated, and loaded (15 μL) onto a 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio Rad, Cat #4568094). SDS-PAGE images are shown in FIG. 2.
b. Expression and Purification of MMLV RTase and Mutant Variants
A colony with the appropriate strain was used to inoculate TB media (1 mL, in a 96-well deep well plate) with kanamycin (0.05 mg/mL) and grown at 37° C. until an OD of approximately 0.9 was achieved followed by cooling of the plate on ice for 5 minutes. Protein expression was induced by the addition of 100 mM IPTG (5 μL), followed by growth at 18° C. for 21 hours. Cells were harvested by spinning samples at 4,700×g for 10 minutes.
Cell pellets were re-suspended in a lysis buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, and 10 mM imidazole) and lysed by the addition of 1× BugBuster® (Millipore Sigma, Cat #70921) and incubation on an end-over-end mixer for 15 minutes at room temperature. Cell debris was removed by centrifuging the lysate at 16,000×g for 20 minutes at 4° C.
Cleared lysates were applied to a HisPur™ Ni-NTA spin plate (ThermoFisher, Cat #88230). Resin was equilibrated with Screening His-Bind buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, and 10 mM imidazole) and samples loaded. Samples were washed three times with Screening His-Wash buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, and 25 mM imidazole) and eluted using Screening His-Elution buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, and 250 mM imidazole). Purified proteins were normalized to a set concentration (100 nM) for testing purposes.
Example 3: Evaluation of Reverse Transcriptase Mutants a. Evaluation of Ability of RTase Mutants to Synthesize DNA
The ability of mutant RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) was compared to an MMLV RTase base construct (RNase H minus construct). Mutant MMLV RTases were tested in two formats: (1) standard two-step cDNA synthesis with gene specific primers, followed by qPCR, and (2) one-step addition of the RTase in Integrated DNA Technologies PrimeTime® Gene Expression Master Mix (GEM).
b. Standard Two-Step Procedure
RTases (2 μL, 100 nM) were added to a reaction mixture containing RNA (50 ng), dNTPs (100 μM), gene specific primer set (500 nM; see Table 2), first strand synthesis buffer (1×, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT), and SuperaseIN (0.17 U/μL) in a 50 μL volume. The reaction was allowed to proceed at 50° C. for 15 minutes, followed by incubation at 80° C. for 10 minutes.
cDNA synthesized by RTase mutants was quantified by qPCR amplification using an assay that identified the SFRS9 gene in human cells. The assay master mix composition included GEM (1×), ROX (50 nM), SFRS9 primer set (500 nM; see Table 2), and SFRS9 probe (250 nM; see Table 2). Assay master mix and synthesized cDNA were mixed at a 4:1 ratio for a final volume of 20 μL. The reaction was run on qPCR (QuantStudio) for 40 cycles under the following cycle conditions: 95° C. hold for 3 minutes, 95° C. for 15 seconds, and 60° C. for one minute.
TABLE 2
Sequences of primers and
probes used for qPCR assays.
SEQ Primer Primer Sequence
ID NO: Name (5′-3′)
633 Hs SFRS9 GTCGAGTATCTCAGAAAAGAAGACA
Forward
Primer
634 Hs SFRS9 CTCGGATGTAGGAAGTTTCACC
Reverse
Primer
635 Hs SFRS9 /5SUN/ATGCCCTGC/
Probe- ZEN/GTAAACTGGATGACA
SUN /3IABkFQ/
c. One-Step Procedure in GEM
RTases (1 μL, 100 nM) were added to a reaction mixture containing RNA (10 ng), GEM (1×), ROX (50 nM), SFRS9 primer set (500 nM; see Table 2), and SFRS9 probe (250 nM; see Table 2) in a final volume of 20 μL. The reaction was run on a qPCR machine (QuantStudio) for 40 cycles using the following cycle conditions: 60° C. hold for 15 minutes, 95° C. hold for 3 minutes, 95° C. for 15 seconds, and 60° C. for one minute.
d. MMLV RTase Base Construct and Single Mutant Variants
As described in Example 1, MMLV RTase single mutant variants were prepared by introducing selected mutations into the MMLV RTase base construct by site-directed mutagenesis, using standard PCR conditions and primers. The sequences of the MMLV RTase base construct and single mutant variants are shown in Table 3. One of skill in the art will understand that the MMLV RTase amino acid sequences set forth in SEQ ID NO: 637 and SEQ ID NO: 717 (the latter of which is described in Example 6 below) are truncated forms of the full-length amino acid sequence of wild-type, or naturally occurring, MMLV RTase. In addition, a person having ordinary skill in the art will understand that a methionine residue is required to recombinantly produce the MMLV RTase base construct and mutants of the disclosure, and as such, that the MMLV RTase sequences disclosed herein (see, e.g., Table 3 below, Table 8 in Example 4, Tables 9 and 12 in Example 5, Table 22 in Example 6, and Table 38 in Example 9) include a methionine residue at the N-terminal end of the amino acid sequence. However, with respect to the present disclosure and for the purpose of identifying and numbering residues in the MMLV RTase amino acid sequence where mutations have been introduced, this methionine residue is considered to be amino acid residue 0 (i.e., is not counted) and the second amino acid residue (e.g., threonine in the MMLV RTase base construct set forth in SEQ ID NO: 637 and SEQ ID NO: 717) is considered to be amino acid residue 1.
TABLE 3
Sequences of MMLV RTase base construct
and single mutant MMLV RTase
constructs.
SEQ
ID Construct Sequence
NO: Construct (DNA: 5′-3′ or AA)
636 MMLV RTase ATGACTTTAAATATTGAGGA
TGAGCATCGTTTACATGAGA
CATCAAAAGAACCCGACGTG
AGCTTAGGGTCAACGTGGCT
TTCTGACTTCCCCCAGGCGT
GGGCGGAGACTGGCGGAATG
GGGTTAGCTGTCCGCCAAGC
ACCGTTGATCATCCCGTTAA
AGGCAACGTCTACACCTGTC
TCTATCAAACAGTACCCCAT
GAGTCAAGAGGCCCGCCTGG
GGATTAAGCCACATATTCAG
CGCTTGCTGGACCAGGGGAT
CTTGGTCCCATGTCAATCTC
CGTGGAACACCCCCCTTCTG
CCCGTGAAAAAGCCAGGTAC
AAACGATTATCGTCCAGTTC
AAGATCTTCGCGAGGTCAAC
AAACGCGTAGAAGACATCCA
TCCGACTGTACCTAATCCTT
ATAATCTGTTATCAGGCCTG
CCCCCATCGCACCAATGGTA
TACAGTATTAGACTTGAAAG
ACGCGTTCTTTTGCCTGCGT
CTGCACCCAACGTCTCAGCC
GCTGTTTGCGTTCGAATGGC
GTGATCCTGAAATGGGAATT
TCGGGTCAGTTAACCTGGAC
TCGTCTGCCCCAGGGCTTTA
AAAACAGCCCCACATTGTTC
GATGAAGCACTTCACCGTGA
CTTAGCAGACTTCCGTATCC
AACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCT
TTTGTTGGCGGCAACGTCTG
AACTTGACTGTCAGCAAGGC
ACACGCGCGTTATTACAAAC
GTTAGGTAACTTAGGATATC
GTGCGTCCGCGAAAAAGGCG
CAAATTTGTCAAAAACAGGT
AAAGTACCTTGGGTATTTGC
TGAAAGAAGGTCAACGTTGG
CTGACTGAAGCGCGTAAGGA
GACCGTAATGGGGCAGCCTA
CGCCTAAGACGCCACGCCAG
TTGCGTGAATTTTTGGGCAC
AGCGGGATTCTGTCGTTTAT
GGATTCCTGGGTTCGCTGAA
ATGGCTGCACCCCTGTACCC
CTTAACAAAAACAGGGACGC
TTTTCAACTGGGGGCCAGAC
CAGCAAAAGGCGTATCAGGA
GATCAAACAAGCTTTGTTGA
CCGCACCCGCGTTGGGTCTT
CCGGATTTAACCAAGCCCTT
TGAGCTGTTCGTTGATGAAA
AACAGGGATATGCAAAAGGA
GTATTAACCCAAAAGTTAGG
CCCGTGGCGTCGCCCTGTTG
CTTACTTGAGTAAAAAATTG
GATCCTGTCGCAGCAGGATG
GCCACCGTGCTTGCGTATGG
TCGCGGCAATTGCCGTTTTG
ACAAAGGATGCAGGTAAGTT
GACGATGGGTCAACCCTTAG
TAATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCA
GCCCCCAGACCGCTGGCTTT
CTAATGCGCGCATGACCCAC
TATCAGGCGCTTCTGCTTGA
TACGGATCGTGTACAATTTG
GACCAGTTGTAGCTTTGAAT
CCAGCTACTTTGCTTCCCCT
TCCAGAAGAAGGACTTCAGC
ACAATTGTTTAGATATTCTG
GCCGAGGCACATGGGACGCG
CCCTGATTTGACGGATCAGC
CACTGCCTGATGCCGACCAT
ACATGGTATACTGGCGGCAG
TAGTCTTCTTCAAGAGGGGC
AACGCAAGGCGGGAGCAGCC
GTCACTACGGAGACCGAAGT
TATCTGGGCCAAAGCGTTAC
CCGCGGGAACATCCGCGCAA
CGTGCACAGTTAATCGCTCT
GACACAGGCCCTGAAGATGG
CAGAGGGCAAAAAGTTGAAT
GTCTACACCAACTCACGTTA
TGCTTTTGCAACAGCGCATA
TCCATGGCGAAATTTACCGC
CGCCGTGGTCTGCTGACTAG
TGAGGGTAAGGAAATTAAAA
ATAAAGATGAGATTCTTGCG
TTGTTAAAAGCTTTATTCTT
ACCAAAACGCCTTTCGATCA
TTCATTGCCCGGGGCATCAA
AAGGGTCACTCAGCGGAGGC
TCGTGGAAACCGTATGGCGG
ACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGA
TACATCAACGCTGTTGATCG
AAAACAGCTCTCCCTACACT
AGCGAGCATTTTTAA
637 MMLV RTase MTLNIEDEHRLHETSKEPDV
SLGSTWLSDFPQAWAETGGM
GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPWALNP
ATLLPLPEEGLQHNCLDILA
EAHGTRPDLTDQPLPDADHT
WYTGGSSLLQEGQRKAGAAV
TTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNV
YTNSRYAFATAHIHGEIYRR
RGLLTSEGKEIKNKDEILAL
LKALFLPKRLSIIHCPGHQK
GHSAEARGNRMADQAARKAA
ITETPDTSTLLIENSSPYTS
EHF
638 MMLV RTase MTLNIEDEHRLHETSKEPDV
with 161R SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SRKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDL
ILLQYVDDLLLAATSELDCQ
QGTRALLQTLGNLGYRASAK
KAQICQKQVKYLGYLLKEGQ
RWLTEARKETVMGQPTPKTP
RQLREFLGTAGFCRLWIPGF
AEMAAPLYPLTKTGTLFNWG
PDQQKAYQEIKQALLTAPAL
GLPDLTKPFELFVDEKQGYA
KGVLTQKLGPWRRPVAYLSK
KLDPVAAGWPPCLRMVAAIA
VLTKDAGKLTMGQPLVILAP
HAVEALVKQPPDRWLSNARM
THYQALLLDTDRVQFGPWAL
NPATLLPLPEEGLQHNCLDI
LAEAHGTRPDLTDQPLPDAD
HTWYTGGSSLLQEGQRKAGA
AVTTETEVIWAKALPAGTSA
QRAQLIALTQALKMAEGKKL
NVYTNSRYAFATAHIHGEIY
RRRGLLTSEGKEIKNKDEIL
ALLKALFLPKRLSIIHCPGH
QKGHSAEARGNRMADQAARK
AAITETPDTSTLLIENSSPY
TSEHF
639 MMLV RTase MTLNIEDEHRLHETSKEPDV
with Q68R SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSREARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPWALNP
ATLLPLPEEGLQHNCLDILA
EAHGTRPDLTDQPLPDADHT
WYTGGSSLLQEGQRKAGAAV
TTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNV
YTNSRYAFATAHIHGEIYRR
RGLLTSEGKEIKNKDEILAL
LKALFLPKRLSIIHCPGHQK
GHSAEARGNRMADQAARKAA
ITETPDTSTLLIENSSPYTS
EHF
640 MMLV RTase MTLNIEDEHRLHETSKEPDV
with Q79R SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIR
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQ
ALLTAPALGLPDLTKPFELF
VDEKQGYAKGVLTQKLGPWR
RPVAYLSKKLDPVAAGWPPC
LRMVAAIAVLTKDAGKLTMG
QPLVILAPHAVEALVKQPPD
RWLSNARMTHYQALLLDTDR
VQFGPWALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLT
DQPLPDADHTWYTGGSSLLQ
EGQRKAGAAVTTETEVIWAK
ALPAGTSAQRAQLIALTQAL
KMAEGKKLNVYTNSRYAFAT
AHIHGEIYRRRGLLTSEGKE
IKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNR
MADQAARKAAITETPDTSTL
LIENSSPYTSEHF
641 MMLV RTase MTLNIEDEHRLHETSKEPDV
with L99R SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPRL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPWALNP
ATLLPLPEEGLQHNCLDILA
EAHGTRPDLTDQPLPDADHT
WYTGGSSLLQEGQRKAGAAV
TTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNV
YTNSRYAFATAHIHGEIYRR
RGLLTSEGKEIKNKDEILAL
LKALFLPKRLSIIHCPGHQK
GHSAEARGNRMADQAARKAA
ITETPDTSTLLIENSSPYTS
EHF
642 MMLV RTase MTLNIEDEHRLHETSKEPDV
with E282D SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQ
ALLLDTDRVQFGPWALNPAT
LLPLPEEGLQHNCLDILAEA
HGTRPDLTDQPLPDADHTWY
TGGSSLLQEGQRKAGAAVTT
ETEVIWAKALPAGTSAQRAQ
LIALTQALKMAEGKKLNVYT
NSRYAFATAHIHGEIYRRRG
LLTSEGKEIKNKDEILALLK
ALFLPKRLSIIHCPGHQKGH
SAEARGNRMADQAARKAAIT
ETPDTSTLLIENSSPYTSEH
F
643 MMLV RTase MTLNIEDEHRLHETSKEPDV
with R298A SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPAQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPWALNP
ATLLPLPEEGLQHNCLDILA
EAHGTRPDLTDQPLPDADHT
WYTGGSSLLQEGQRKAGAAV
TTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNV
YTNSRYAFATAHIHGEIYRR
RGLLTSEGKEIKNKDEILAL
LKALFLPKRLSIIHCPGHQK
GHSAEARGNRMADQAARKAA
ITETPDTSTLLIENSSPYTS
EHF
e. Experimental Results
The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase single mutant variants were analyzed and reported by copy number output based on a standard curve (see Tables 4 and 5). Six single mutant MMLV RTase variants were found to exhibit an increase in the overall activity and thermostability as compared to the MMLV RTase base construct. The six single mutant MMLV RTase variants were as follows: I61R, Q68R, Q79R, L99R, E282D, and R298A.
TABLE 4
Two-step cDNA synthesis by MMLV RT single mutants.
Data was generated via qPCR human normalizer
assay and translated by copy number.
MMLV RT Variant Quantity Mean Quantity Standard Deviation
MMLV-II 21,046.784 954.827
MMLV-II A283V 280.423 50.910
MMLV-II A283R 10,390.819 340.236
MMLV-II A283E 7,378.705 122.716
MMLV-II E123A 15,059.791 556.095
MMLV-II E123R 19,043.292 415.522
MMLV-II E123D 3,619.959 243.766
MMLV-II E282A 19,939.551 1,645.246
MMLV-II E282R 15,588.940 546.467
MMLV-II E282D 24,282.327 2,259.264
MMLV-II I61A 648.252 45.640
MMLV-II I61R 26,280.811 549.417
MMLV-II I61E 10,966.741 469.747
MMLV-II K102A 98.438 12.778
MMLV-II K102R 780.114 90.331
MMLV-II K102E 1,674.854 157.485
MMLV-II K103A 359.984 67.322
MMLV-II K103R 206.765 20.758
MMLV-II K103E 200.883 16.719
MMLV-II K120A 217.787 72.696
MMLV-II K120R 3,619.338 100.478
MMLV-II K120E 2,230.375 210.050
MMLV-II K193A 2,736.271 162.383
MMLV-II K193R 11,496.935 193.681
MMLV-II K193E 325.109 50.932
MMLV-II K295A 8,101.927 348.373
MMLV-II K295R 6,879.112 131.993
MMLV-II K295E 9,673.612 351.106
MMLV-II K329A 3,199.167 212.003
MMLV-II K329R 10,387.670 330.429
MMLV-II K329E 18,306.813 1,167.600
MMLV-II K53A 474.465 62.390
MMLV-II K53R 369.020 49.436
MMLV-II K53E 5,308.165 104.585
MMLV-II K62A 2,102.396 64.197
MMLV-II K62R 4,920.330 251.414
MMLV-II K62E 71.723 11.419
MMLV-II K75A 76.659 24.657
MMLV-II K75R 2,842.314 77.212
MMLV-II K75E 1,697.887 158.946
MMLV-II L99A 1,576.246 213.455
MMLV-II L99R 37,070.048 1,531.910
MMLV-II L99E 195.448 22.530
MMLV-II N107A 3,354.325 176.385
MMLV-II N107R 41.532 24.527
MMLV-II N107E 8,523.285 353.411
MMLV-II Q291A 14,093.444 576.318
MMLV-II Q291R 15,736.443 566.630
MMLV-II Q291E 1,480.309 93.187
MMLV-II Q68A n.d. n.d.
MMLV-II Q68R 20,158.035 722.022
MMLV-II Q68E 2,263.714 150.236
MMLV-II Q79A 2,317.484 43.518
MMLV-II Q79R 37,480.443 1,268.309
MMLV-II Q79E 489.184 39.449
MMLV-II R110A 1,815.710 7.917
MMLV-II R110K 502.172 38.619
MMLV-II R110E 383.331 38.162
MMLV-II R298A 44,477.013 3,036.502
MMLV-II R298K 14,925.202 186.581
MMLV-II R298E 1,150.932 56.107
MMLV-II R301A 2,745.075 82.646
MMLV-II R301K 12,813.899 568.898
MMLV-II R301E 1,583.826 198.913
MMLV-II T106A 16,641.642 179.631
MMLV-II T106R 2,248.217 71.295
MMLV-II T106E 10,302.113 250.531
MMLV-II T128V 7,034.032 351.446
MMLV-II T128R 3,465.069 143.456
MMLV-II T128E 10,709.019 110.124
MMLV-II T293A 4,612.880 167.335
MMLV-II T293R 13,753.879 319.851
MMLV-II T293E 12,893.457 223.100
MMLV-II T296A 2,192.531 76.071
MMLV-II T296R 893.449 51.913
MMLV-II T296E 473.936 102.414
MMLV-II T55A 5,774.471 223.173
MMLV-II T55R 3,284.089 314.651
MMLV-II T55E 6,143.058 429.507
MMLV-II T57A 6,129.791 285.070
MMLV-II T57R 888.244 11.952
MMLV-II T57E 1,487.448 71.681
MMLV-II V101A 552.130 98.391
MMLV-II V101R 4,754.017 107.434
MMLV-II V101E 1,388.699 87.091
MMLV-II V112A 2,085.594 72.265
MMLV-II V112R 377.194 41.722
MMLV-II V112E 210.825 17.715
MMLV-II V59A 628.779 15.216
MMLV-II V59R 6,662.173 210.234
MMLV-II V59E 3,249.465 79.848
MMLV-II Y109A 101.656 6.717
MMLV-II Y109R 349.373 27.171
MMLV-II Y109E 1,029.589 45.189
MMLV-IV 71,572.714 4,656.679
TABLE 5
One-step cDNA synthesis by MMLV RT single mutants.
Data was generated via qPCR human normalizer assay
and data is translated by copy number.
MMLV RT Variant Quantity Mean Quantity Standard Deviation
MMLV-II 20,638.973 614.785
MMLV-II A283V 8,802.753 220.902
MMLV-II A283R 14,379.575 337.562
MMLV-II A283E 16,396.614 203.476
MMLV-II E123A 17,975.218 259.986
MMLV-II E123R 20,652.508 515.600
MMLV-II E123D 14,452.672 242.000
MMLV-II E282A 19,017.751 827.419
MMLV-II E282R 17,180.421 204.739
MMLV-II E282D 20,735.271 420.881
MMLV-II I61A 7,450.147 348.788
MMLV-II I61R 25,123.507 2,977.836
MMLV-II I61E 17,441.860 1,662.749
MMLV-II K102A 9,342.754 120.846
MMLV-II K102R 10,563.589 255.139
MMLV-II K102E 13,925.008 307.601
MMLV-II K103A 9,429.555 437.351
MMLV-II K103R 9,009.846 155.888
MMLV-II K103E 7,985.278 189.792
MMLV-II K120A 8,593.433 438.722
MMLV-II K120R 12,558.793 407.946
MMLV-II K120E 12,268.574 303.495
MMLV-II K193A 12,977.263 537.992
MMLV-II K193R 13,446.766 2,337.906
MMLV-II K193E 8,536.558 182.514
MMLV-II K295A 13,506.491 1,613.467
MMLV-II K295R 13,944.407 1,839.608
MMLV-II K295E 15,021.823 650.111
MMLV-II K329A 13,284.541 246.298
MMLV-II K329R 15,935.899 970.971
MMLV-II K329E 20,628.859 884.254
MMLV-II K53A 10,868.676 161.435
MMLV-II K53R 9,908.252 632.663
MMLV-II K53E 20,666.775 518.895
MMLV-II K62A 9,454.043 732.242
MMLV-II K62R 14,532.171 63.450
MMLV-II K62E 8,341.361 436.076
MMLV-II K75A 9,084.502 113.100
MMLV-II K75R 13,106.462 331.663
MMLV-II K75E 11,191.849 565.160
MMLV-II L99A 12,876.076 49.507
MMLV-II L99R 27,167.197 142.371
MMLV-II L99E 6,534.199 2,730.598
MMLV-II N107A 13,563.421 349.378
MMLV-II N107R 8,654.167 497.167
MMLV-II N107E 16,675.075 172.596
MMLV-II Q291A 20,957.729 150.006
MMLV-II Q291R 17,980.723 346.436
MMLV-II Q291E 11,025.722 407.116
MMLV-II Q68A n.d. n.d.
MMLV-II Q68R 24,925.791 937.265
MMLV-II Q68E 12,844.484 165.039
MMLV-II Q79A 12,038.975 482.596
MMLV-II Q79R 28,458.521 296.595
MMLV-II Q79E 10,358.863 309.043
MMLV-II R110A 11,517.764 562.094
MMLV-II R110K 8,112.167 76.742
MMLV-II R110E 8,809.423 290.785
MMLV-II R298A 27,817.905 172.690
MMLV-II R298K 18,222.660 825.743
MMLV-II R298E 10,783.790 783.279
MMLV-II R301A 11,344.854 63.499
MMLV-II R301K 17,584.850 445.587
MMLV-II R301E 10,146.906 1,879.902
MMLV-II T106A 17,717.520 215.965
MMLV-II T106R 11,680.187 148.213
MMLV-II T106E 21,203.557 366.469
MMLV-II T128V 14,384.970 355.754
MMLV-II T128R 12,938.223 464.841
MMLV-II T128E 14,781.394 1,930.931
MMLV-II T293A 15,658.189 347.640
MMLV-II T293R 19,976.165 253.604
MMLV-II T293E 17,580.335 404.397
MMLV-II T296A 10,312.142 159.775
MMLV-II T296R 8,482.071 92.806
MMLV-II T296E 7,687.972 112.884
MMLV-II T55A 18,073.262 618.174
MMLV-II T55R 11,546.179 138.906
MMLV-II T55E 12,299.658 815.911
MMLV-II T57A 14,700.042 2,916.521
MMLV-II T57R 11,195.901 145.433
MMLV-II T57E 11,958.503 605.445
MMLV-II V101A 10,697.751 269.696
MMLV-II VI01R 8,934.765 53.924
MMLV-II V101E 11,295.874 296.506
MMLV-II V112A 12,854.738 356.724
MMLV-II V112R 6,331.802 303.453
MMLV-II V112E 7,643.184 448.446
MMLV-II V59A 9,520.143 339.954
MMLV-II V59R 18,523.053 499.377
MMLV-II V59E 16,029.631 137.454
MMLV-II Y109A 8,421.361 185.196
MMLV-II Y109R 8,581.961 129.732
MMLV-II Y109E 10,216.473 416.388
MMLV-IV 65,726.159 1,811.314
Example 4: Extension of Reverse Transcriptase Single Mutants The amino acid positions that enclosed the MMLV RTase single mutants identified in Example 3 were further evaluated to include all possible amino acid substitutions at that position. The single mutants were cloned, overexpressed, and purified as described in Examples 1 and 2, and evaluated as described in Example 3. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase double mutant variants were analyzed and reported by copy number output based on a standard curve (see Tables 6 and 7). Ten single mutant MMLV RTase variants (see Table 8) were found to exhibit an increase in the overall activity and thermostability as compared to the MMLV RTase base construct. The ten single mutant MMLV RTase variants were as follows: I61K, I61M, Q68I, Q68K, Q79H, Q79I, L99K, L99N, E282M and E282W.
TABLE 6
Two-step cDNA synthesis by MMLV RT single mutants.
Data was generated via qPCR human normalizer
assay and translated by copy number.
MMLV RT Variant Quantity Mean Quantity Standard Deviation
MMLV-II 1,484.121 125.278
MMLV-II E282C 749.332 37.947
MMLV-II E282F 968.042 28.112
MMLV-II E282G 841.839 30.618
MMLV-II E282H 936.562 64.904
MMLV-II E282I 1,418.551 8.682
MMLV-II E282K 2,399.973 50.862
MMLV-II E282L 1,778.903 134.133
MMLV-II E282M 2,115.328 125.477
MMLV-II E282N 1,175.130 79.221
MMLV-II E282P 1,529.331 61.525
MMLV-II E282Q 1,856.418 24.118
MMLV-II E282S 673.670 44.770
MMLV-II E282T 994.318 24.066
MMLV-II E282V 748.877 29.053
MMLV-II E282W 2,469.404 141.080
MMLV-II E282Y 1,360.706 338.309
MMLV-II I61C 283.240 11.244
MMLV-II I61D 349.008 10.979
MMLV-II I61F 784.163 22.643
MMLV-II I61G 395.348 21.967
MMLV-II I61H 736.015 30.271
MMLV-II I61K 4,479.606 62.627
MMLV-II I61L 1,106.547 38.553
MMLV-II I61M 4,198.088 93.025
MMLV-II I61N 709.752 29.312
MMLV-II I61P 32.935 16.814
MMLV-II I61Q 1,311.695 145.810
MMLV-II I61S 797.783 50.626
MMLV-II I61T 628.173 33.371
MMLV-II I61V 1,439.915 27.490
MMLV-II I61W 442.039 29.310
MMLV-II I61Y 534.249 26.831
MMLV-II L99C 3,109.142 80.016
MMLV-II L99D 83.653 3.432
MMLV-II L99F 2,811.513 79.584
MMLV-II L99G 908.041 16.157
MMLV-II L99H 4,881.196 390.497
MMLV-II L99I 910.072 71.671
MMLV-II L99K 6,410.818 127.262
MMLV-II L99M 976.548 65.154
MMLV-II L99N 4,974.458 162.464
MMLV-II L99P 6.416 1.820
MMLV-II L99Q 3,908.473 337.167
MMLV-II L99S 3,793.955 86.959
MMLV-II L99T 4,189.211 27.640
MMLV-II L99V 964.081 48.105
MMLV-II L99W 1,614.660 40.442
MMLV-II L99Y 2,123.406 181.945
MMLV-II Q68A 1,184.702 7.676
MMLV-II Q68C 2,038.167 36.463
MMLV-II Q68D 1,613.880 77.796
MMLV-II Q68F 1,805.647 62.456
MMLV-II Q68G 2,262.873 69.688
MMLV-II Q68H 106.421 9.860
MMLV-II Q68I 2,675.446 73.874
MMLV-II Q68K 1,042.979 70.081
MMLV-II Q68L 1,070.742 57.215
MMLV-II Q68M 1,342.806 58.349
MMLV-II Q68N 1,993.946 65.808
MMLV-II Q68P 2,025.753 25.540
MMLV-II Q68S 1,895.984 26.959
MMLV-II Q68T 431.442 22.751
MMLV-II Q68V 1,534.710 110.794
MMLV-II Q68W 1,790.706 124.583
MMLV-II Q79C 2,477.812 107.510
MMLV-II Q79D 627.902 11.073
MMLV-II Q79F 1,786.571 126.904
MMLV-II Q79G 2,702.985 83.998
MMLV-II Q79H 2,851.710 57.501
MMLV-II Q79I 2,967.710 57.440
MMLV-II Q79K 1,346.751 64.513
MMLV-II Q79L 2,214.615 67.622
MMLV-II Q79M 1,847.181 31.384
MMLV-II Q79N 1,365.563 54.775
MMLV-II Q79P 674.074 42.100
MMLV-II Q79S 2,199.353 52.958
MMLV-II Q79T 1,523.163 77.025
MMLV-II Q79V 1,704.661 77.643
MMLV-II Q79W 2,186.489 31.470
MMLV-II Q79Y 2,326.023 123.508
MMLV-II R298C 79.970 9.815
MMLV-II R298D 0.000 0.000
MMLV-II R298F 84.760 9.362
MMLV-II R298G 357.027 15.726
MMLV-II R298H 269.257 20.814
MMLV-II R298I 130.983 5.364
MMLV-II R298L 199.612 5.843
MMLV-II R298M 172.013 18.710
MMLV-II R298N 199.678 2.660
MMLV-II R298P 122.098 5.900
MMLV-II R298Q 118.092 40.694
MMLV-II R298S 406.112 7.695
MMLV-II R298T 618.616 20.023
MMLV-II R298V 136.498 13.297
MMLV-II R298W 68.096 7.016
MMLV-II R298Y 162.713 7.854
MMLV-IV 6,830.294 376.878
TABLE 7
One-step cDNA synthesis by MMLV RT single mutants.
Data was generated via qPCR human normalizer assay
and data is translated by copy number.
MMLV RT Variant Quantity Mean Quantity Standard Deviation
MMLV-II 408.018 8.693
MMLV-II E282C 175.083 7.005
MMLV-II E282F 1,043.025 16.137
MMLV-II E282G 635.037 13.293
MMLV-II E282H 656.956 10.018
MMLV-II E282I 1,033.125 44.996
MMLV-II E282K 751.309 17.611
MMLV-II E282L 1,072.350 80.365
MMLV-II E282M 1,318.072 51.735
MMLV-II E282N 539.305 10.767
MMLV-II E282P 725.869 92.685
MMLV-II E282Q 626.674 12.129
MMLV-II E282S 354.956 34.850
MMLV-II E282T 485.477 45.783
MMLV-II E282V 594.047 27.898
MMLV-II E282W 913.290 61.145
MMLV-II E282Y 759.920 34.784
MMLV-II I61C 219.438 18.403
MMLV-II I61D 347.020 13.303
MMLV-II I61F 428.623 25.316
MMLV-II I61G 389.503 21.764
MMLV-II I61H 514.330 18.416
MMLV-II I61K 2,343.894 67.214
MMLV-II I61L 621.572 14.892
MMLV-II I61M 2,536.807 150.371
MMLV-II I61N 538.519 20.736
MMLV-II I61P 61.683 18.802
MMLV-II I61Q 701.471 32.487
MMLV-II I61S 611.977 30.430
MMLV-II I61T 534.254 31.643
MMLV-II I61V 881.608 20.662
MMLV-II I61W 428.440 17.964
MMLV-II I61Y 347.930 4.412
MMLV-II L99C 2,390.104 35.867
MMLV-II L99D 185.044 6.975
MMLV-II L99F 1,577.767 7.757
MMLV-II L99G 987.225 9.718
MMLV-II L99H 3,886.372 111.670
MMLV-II L99I 613.648 46.303
MMLV-II L99K 7,597.650 321.753
MMLV-II L99M 934.817 52.006
MMLV-II L99N 4,689.222 160.641
MMLV-II L99P 18.537 1.131
MMLV-II L99Q 2,394.744 64.077
MMLV-II L99S 3,293.831 111.802
MMLV-II L99T 3,505.113 101.670
MMLV-II L99V 677.756 49.356
MMLV-II L99W 839.088 50.301
MMLV-II L99Y 1,127.536 19.074
MMLV-II Q68A 827.617 30.689
MMLV-II Q68C 1,110.680 45.944
MMLV-II Q68D 1,045.802 25.488
MMLV-II Q68F 1,210.166 120.899
MMLV-II Q68G 907.279 30.688
MMLV-II Q68H 150.384 6.867
MMLV-II Q68I 1,550.372 76.712
MMLV-II Q68K 1,712.176 47.342
MMLV-II Q68L 651.039 51.426
MMLV-II Q68M 1,395.463 34.805
MMLV-II Q68N 1,241.364 25.780
MMLV-II Q68P 1,249.444 13.709
MMLV-II Q68S 1,125.260 21.324
MMLV-II Q68T 792.901 31.513
MMLV-II Q68V 1,026.654 24.972
MMLV-II Q68W 1,594.175 101.221
MMLV-II Q79C 1,948.151 87.341
MMLV-II Q79D 458.131 10.763
MMLV-II Q79F 1,623.675 50.723
MMLV-II Q79G 1,885.097 20.190
MMLV-II Q79H 2,508.763 149.926
MMLV-II Q79I 2,329.030 76.545
MMLV-II Q79K 1,861.302 24.320
MMLV-II Q79L 1,496.247 30.399
MMLV-II Q79M 1,496.469 38.178
MMLV-II Q79N 995.813 42.279
MMLV-II Q79P 526.914 23.216
MMLV-II Q79S 1,685.124 42.694
MMLV-II Q79T 966.505 8.377
MMLV-II Q79V 1,218.191 21.512
MMLV-II Q79W 1,962.326 37.135
MMLV-II Q79Y 2,218.504 56.938
MMLV-II R298C 45.500 1.456
MMLV-II R298D 0.000 0.000
MMLV-II R298F 104.825 5.133
MMLV-II R298G 323.542 14.052
MMLV-II R298H 253.202 47.711
MMLV-II R298I 205.982 8.304
MMLV-II R298L 213.674 15.199
MMLV-II R298M 176.347 12.484
MMLV-II R298N 142.969 39.198
MMLV-II R298P 188.995 3.689
MMLV-II R298Q 95.525 44.292
MMLV-II R298S 307.614 9.962
MMLV-II R298T 487.828 3.480
MMLV-II R298V 255.828 12.902
MMLV-II R298W 37.872 8.482
MMLV-II R298Y 153.333 25.137
MMLV-IV 19,407.721 466.310
TABLE 8
Sequences of single mutant MMLV
RTase variants.
SEQ
ID
NO: Construct Construct Sequence (AA)
644 MMLV RTase MTLNIEDEHRLHETSKEPDV
with 161K SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SKKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
645 MMLV RTase MTLNIEDEHRLHETSKEPDV
with 161M SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SMKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
646 MMLV RTase MTLNIEDEHRLHETSKEPDV
with Q68I SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSIEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
647 MMLV RTase MTLNIEDEHRLHETSKEPDV
with Q68K SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSKEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
648 MMLV RTase MTLNIEDEHRLHETSKEPDV
with Q79H SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIH
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
649 MMLV RTase MTLNIEDEHRLHETSKEPDV
with Q79I SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHII
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
650 MMLV RTase MTLNIEDEHRLHETSKEPDV
with L99K SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTP
VSIKQYPMSQEA
RLGIKPHIQRLLDQGILVPC
QSPWNTPLKPVKKPGTNDYR
PVQDLREVNKRVEDIHPTVP
NPYNLLSGLPPSHQWYTVLD
LKDAFFCLRLHPTSQPLFAF
EWRDPEMGISGQLTWTRLPQ
GFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNL
GYRASAKKAQICQKQVKYLG
YLLKEGQRWLTEARKETVMG
QPTPKTPRQLREFLGTAGFC
RLWIPGFAEMAAPLYPLTKT
GTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFV
DEKQGYAKGVLTQKLGPWRR
PVAYLSKKLDPVAAGWPPCL
RMVAAIAVLTKDAGKLTMGQ
PLVILAPHAVEALVKQPPDR
WLSNARMTHYQALLLDTDRV
QFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLT
DQPLPDADHTWYTGGSSLLQ
EGQRKAGAAVTTETEVIWAK
ALPAGTSAQRAQLIALTQAL
KMAEGKKLNVYTNSRYAFAT
AHIHGEIYRRRGLLTSEGKE
IKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNR
MADQAARKAAITETPDTSTL
LIENSSPYTSEHF
651 MMLV RTase MTLNIEDEHRLHETSKEPDV
with L99N SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLN
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
652 MMLV RTase MTLNIEDEHRLHETSKEPDV
with E282M SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTMARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
653 MMLV RTase MTLNIEDEHRLHETSKEPDV
with E282W SLGSTWLSDFPQAWAETGGM
mutation GLAVRQAPLIIPLKATSTPV
SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTWARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
Example 5: Stacking of Reverse Transcriptase Mutants with Enhanced Activity a. MMLV RTase Double Mutants
The MMLV RTase single mutants identified in Example 3 were stacked to further improve the ability of MMLV RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) as compared to the MMLV RTase base construct (RNase H minus construct). Fifteen MMLV RTase double mutant variants (see Table 9) were cloned, overexpressed, and purified as described in Examples 1 and 2, and evaluated as described in Example 3. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase double mutant variants were analyzed and reported by copy number output based on a standard curve (see Tables 10 and 11).
Four of the fifteen MMLV RTase double mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase double mutant variants, and almost all of the MMLV RTase double mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The four MMLV RTase double mutant variants that were found to exhibit the highest overall activity were E282D/L99R, L99R/Q68R, L99R/Q79R, and Q68R/Q79R.
TABLE 9
Sequences of double mutant
MMLV RTase variants.
SEQ
ID
NO: Construct Construct Sequence (AA)
654 MMLV RTase MTLNIEDEHRLHETSKEPDV
With SLGSTWLSDFPQAWAETGGM
I61R/E282D GLAVRQAPLIIPLKATSTPV
mutations SRKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
655 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
L99R/E282D GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPRL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
656 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/E282D GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSREARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
657 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q79R/E282D GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSQEARLGIKPHIR
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
658 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
E282D/R298A GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPAQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
659 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
I61R/L99R GLAVRQAPLIIPLKATSTPV
mutations SRKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLR
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
660 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
I61R/Q68R GLAVRQAPLIIPLKATSTPV
mutations SRKQYPMSREARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEH
661 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
I61R/Q79R GLAVRQAPLIIPLKATSTPV
mutations SRKQYPMSQEARLGIKPHIR
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
662 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
I61R/R298A GLAVRQAPLIIPLKATSTPV
mutations SRKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPAQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
663 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/L99R GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSREARLGIKPHIQ
RLLDQGILVPCQSPWNTPLR
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
664 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q79R/L99R GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSQEARLGIKPHIR
RLLDQGILVPCQSPWNTPLR
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
665 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
L99R/R298A GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSQEARLGIKPHIQ
RLLDQGILVPCQSPWNTPLR
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPAQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
666 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/Q79R GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSREARLGIKPHIR
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
667 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/R298A GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSREARLGIKPHIQ
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPAQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
668 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q79R/R298A GLAVRQAPLIIPLKATSTPV
mutations SIKQYPMSQEARLGIKPHIR
RLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPAQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTKPFELF
VDEKQGYAKGVLTQKLGPWR
RPVAYLSKKLDPVAAGWPPC
LRMVAAIAVLTKDAGKLTMG
QPLVILAPHAVEALVKQPPD
RWLSNARMTHYQALLLDTDR
VQFGPVVALNPATLLPLPEE
GLQHNCLDILAEAHGTRPDL
TDQPLPDADHTWYTGGSSLL
QEGQRKAGAAVTTETEVIWA
KALPAGTSAQRAQLIALTQA
LKMAEGKKLNVYTNSRYAFA
TAHIHGEIYRRRGLLTSEGK
EIKNKDEILALLKALFLPKR
LSIIHCPGHQKGHSAEARGN
RMADQAARKAAITETPDTST
LLIENSSPYTSEHFTAPALG
LPDL
TABLE 10
Two-Step cDNA synthesis by MMLV RT double mutants.
Data was generated via qPCR human normalizer assay
and data is translated by copy number.
MMLV RT Variant Quantity Mean Quantity Standard Deviation
MMLV-II 1,773.623 5.057
MMLV-II E282D/I61R 4,810.277 143.422
MMLV-II E282D/L99R 7,266.281 50.730
MMLV-II E282D/Q68R 5,186.392 69.563
MMLV-II E282D/Q79R 4,311.403 95.402
MMLV-II E282D/R298A 1,366.524 16.429
MMLV-II I61R/L99R 6,061.812 174.619
MMLV-II I61R/Q68R 5,899.316 39.879
MMLV-II I61R/Q79R 5,257.089 98.378
MMLV-II I61R/R298A 2,661.223 68.948
MMLV-II L99R/Q68R 7,750.519 94.408
MMLV-II L99R/Q79R 7,455.203 124.095
MMLV-II L99R/R298A 5,351.021 179.558
MMLV-II Q68R/Q79R 7,178.681 86.595
MMLV-II Q68R/R298A 4,524.340 84.703
MMLV-II Q79R/R298A 3,739.608 58.621
MMLV-IV 8,258.715 79.458
TABLE 11
One-Step cDNA synthesis by MMLV RT double mutants.
Data was generated via qPCR human normalizer assay
and data is translated by cony number.
MMLV-RT Variant Quantity Mean Quantity Standard Deviation
MMLV-II 859.127 24.795
MMLV-II E282D/I61R 2,948.906 49.177
MMLV-II E282D/L99R 4,814.957 239.110
MMLV-II E282D/Q68R 3,709.046 131.434
MMLV-II E282D/Q79R 3,694.187 98.772
MMLV-II E282D/R298A 794.643 39.913
MMLV-II I61R/L99R 3,443.713 180.210
MMLV-II I61R/Q68R 3,525.138 112.288
MMLV-II I61R/Q79R 3,125.990 120.996
MMLV-II I61R/R298A 2,006.208 83.559
MMLV-II L99R/Q68R 6,755.852 102.788
MMLV-II L99R/Q79R 6,709.502 35.997
MMLV-II L99R/R298A 2,128.451 55.565
MMLV-II Q68R/Q79R 6,343.821 140.779
MMLV-II Q68R/R298A 2,406.470 74.117
MMLV-II Q79R/R298A 2,301.759 22.849
MMLV-IV 15,411.857 333.388
b. Cloning of MMLV RTase Triple and More Mutants
Following the double mutant variants, MMLV RTase single mutants were stacked further to improve the ability of MMLV RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) as compared to the MMLV RTase base construct (RNase H minus construct). Seventeen MMLV RTase triple or more mutant variants (see Table 12) were cloned as described in Example 1.
TABLE 12
Sequences of triple or more mutant
MMLV RTase variants.
SEQ
ID Construct Sequence
NO: Construct (AA)
669 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
L99R/ SIKQYPMSREARLGIKPHIQ
E282D RLLDQGILVPCQSPWNTPLR
mutations PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
670 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q79R/ GLAVRQAPLIIPLKATSTPV
L99R/ SIKQYPMSQEARLGIKPHIR
E282D RLLDQGILVPCQSPWNTPLR
mutations PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
671 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSREARLGIKPHIR
E282D RLLDQGILVPCQSPWNTPLL
mutations PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
672 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSREARLGIKPHIR
L99R RLLDQGILVPCQSPWNTPLR
mutations PVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
673 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSREARLGIKPHIR
L99R/ RLLDQGILVPCQSPWNTPLR
E282D PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
674 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSREARLGIKPHIR
L99K/ RLLDQGILVPCQSPWNTPLK
E282D PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
675 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSREARLGIKPHIR
L99N/ RLLDQGILVPCQSPWNTPLN
E282D PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
676 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68I/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSIEARLGIKPHIR
L99R/ RLLDQGILVPCQSPWNTPLR
E282D PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
677 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68K/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSKEARLGIKPHIR
L99R/ RLLDQGILVPCQSPWNTPLR
E282D PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
678 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79H/ SIKQYPMSREARLGIKPHIH
L99R/ RLLDQGILVPCQSPWNTPLR
E282D PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
679 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79I/ SIKQYPMSREARLGIKPHII
L99R/ RLLDQGILVPCQSPWNTPLR
E282D PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
680 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSREARLGIKPHIR
L99R/ RLLDQGILVPCQSPWNTPLR
E282M PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTMARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
681 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68R/ GLAVRQAPLIIPLKATSTPV
Q79R/ SIKQYPMSREARLGIKPHIR
L99R/ RLLDQGILVPCQSPWNTPLR
E282W PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTWARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
682 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
161K/ GLAVRQAPLIIPLKATSTPV
Q68R/ SKKQYPMSREARLGIKPHIR
Q79R/ RLLDQGILVPCQSPWNTPLR
L99R/ PVKKPGTNDYRPVQDLREVN
E282D KRVEDIHPTVPNPYNLLSGL
mutations PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
683 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
161M/ GLAVRQAPLIIPLKATSTPV
Q68R/ SMKQYPMSREARLGIKPHIR
Q79R/ RLLDQGILVPCQSPWNTPLR
L99R/ PVKKPGTNDYRPVQDLREVN
E282D KRVEDIHPTVPNPYNLLSGL
mutations PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTDARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDL
TKPFELFVDEKQGYAKGVLT
QKLGPWRRPVAYLSKKLDPV
AAGWPPCLRMVAAIAVLTKD
AGKLTMGQPLVILAPHAVEA
LVKQPPDRWLSNARMTHYQA
LLLDTDRVQFGPVVALNPAT
LLPLPEEGLQHNCLDILAEA
HGTRPDLTDQPLPDADHTWY
TGGSSLLQEGQRKAGAAVTT
ETEVIWAKALPAGTSAQRAQ
LIALTQALKMAEGKKLNVYT
NSRYAFATAHIHGEIYRRRG
LLTSEGKEIKNKDEILALLK
ALFLPKRLSIIHCPGHQKGH
SAEARGNRMADQAARKAAIT
ETPDTSTLLIENSSPYTSEH
F
684 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
Q68I/ GLAVRQAPLIIPLKATSTPV
Q79H/ SIKQYPMSIEARLGIKPHIH
L99K/ RLLDQGILVPCQSPWNTPLK
E282M PVKKPGTNDYRPVQDLREVN
mutations KRVEDIHPTVPNPYNLLSGL
PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTMARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
685 MMLV RTase MTLNIEDEHRLHETSKEPDV
with SLGSTWLSDFPQAWAETGGM
I61M/ GLAVRQAPLIIPLKATSTPV
Q68I/ SMKQYPMSIEARLGIKPHIH
Q79H/ RLLDQGILVPCQSPWNTPLK
L99K/ PVKKPGTNDYRPVQDLREVN
E282M KRVEDIHPTVPNPYNLLSGL
mutations PPSHQWYTVLDLKDAFFCLR
LHPTSQPLFAFEWRDPEMGI
SGQLTWTRLPQGFKNSPTLF
DEALHRDLADFRIQHPDLIL
LQYVDDLLLAATSELDCQQG
TRALLQTLGNLGYRASAKKA
QICQKQVKYLGYLLKEGQRW
LTMARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAE
MAAPLYPLTKTGTLFNWGPD
QQKAYQEIKQALLTAPALGL
PDLTKPFELFVDEKQGYAKG
VLTQKLGPWRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVL
TKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTH
YQALLLDTDRVQFGPVVALN
PATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADH
TWYTGGSSLLQEGQRKAGAA
VTTETEVIWAKALPAGTSAQ
RAQLIALTQALKMAEGKKLN
VYTNSRYAFATAHIHGEIYR
RRGLLTSEGKEIKNKDEILA
LLKALFLPKRLSIIHCPGHQ
KGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYT
SEHF
c. Expression and Purification of MMLV RTase and Mutant Variants
A colony with the appropriate strain was used to inoculate TB media (200 mL) with kanamycin (0.05 mg/mL) and grown at 37° C. until an OD of approximately 0.9 was achieved followed by cooling of the flask for 30 minutes at 4° C. Protein expression was induced by the addition of 1 M IPTG (100 μL), followed by growth at 18° C. for 21 hours. Cells were harvested by spinning samples at 4,700×g for 10 minutes.
Cell pellets were re-suspended in a lysis buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 300 mM NaCl, 10 mM imidazole, 5 mM DTT, 0.01% n-ocyl-β-D-glucopyranoside, DNaseI, 10 mM CaCl2), lysozyme (1 mg/mL), and protease inhibitor). The sample was lysed on an Avestin Emulsiflex C3 pre-chilled to 4° C. at 15-20 kpsi with three passes. Cell debris was removed by centrifuging the lysate at 16,000×g for 30 minutes at 4° C.
Cleared lysates were applied to a HisTrap HP column (Cytiva Life Sciences, Cat #17524701). The resin was equilibrated with MMLV His-Bind buffer (50 mM NaPO4, pH 7.8, 5% glycerol, 0.3 M NaCl, 10 mM imidazole, 1 mM DTT and 0.01% IGEPAL-CA), followed by sample loading. The samples were washed with MMLV His-Bind buffer, followed by a 25% B wash (B=MMLV His Elution buffer=50 mM NaPO4, pH 7.8, 5% glycerol, 0.3 M NaCl, 250 mM imidazole, 1 mM DTT and 0.01% IGEPAL-CA). The sample was eluted with 100% B for 10 CVs in 45 mL fractions.
Purified proteins were applied to a HiTrap Heparin HP column (Cytiva Life Sciences, Cat #17040601). The resin was equilibrated with MMLV Heparin-Bind buffer (50 mM Tris HCl pH 8.5, 75 mM NaCl, 1 mM DTT, 5% glycerol and 0.01% IGEPAL-CA), followed by sample loading. The sample was washed with MLV Heparin Bind buffer, followed by a 25% B wash (B=MLV Heparin Elution Buffer). The sample was eluted with 60% B for 10 CVs in 45 mL fractions.
Purified proteins were applied to a Bio-Scale™ Mini CHT™ Cartridge (Bio-Rad Laboratories, Cat #7324322). The resin was washed with 1 M NaOH, followed by equilibration with MMLV Heparin-Bind buffer and sample loading. The sample was washed with MLV Heparin Elution buffer, followed by MMLV Heparin Bind buffer. The sample was linearly eluted to 100% B2 (B2=MMLV HA Elution Buffer=250 mM KPO4 pH 7.5, 1 mM DTT, 5% glycerol and 0.01% IGEPAL-CA) for 15 CVs in 5 mL fractions.
Fractions containing purified protein were pooled and dialyzed in MMLV Storage Buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 50% (v/v) glycerol).
d. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA by Gene Specific Priming
MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Temperatures were adjusted for both two-step and one-step reactions to 55 and 60° C., respectively. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see Tables 13 and 14).
Six of the seventeen MMLV RTase triple or more mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase stacked mutant variants, and almost all of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The six MMLV RTase mutant variants that were found to exhibit the highest overall activity were Q68R/L99R, Q68R/Q79R/L99R, Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99K/E282D, Q68R/Q79R/L99R/E282W, I61M/Q68R/Q79R/L99R/E282D and Q68I/Q79H/L99K/E282M.
TABLE 13
Two-Step cDNA synthesis by MMLV RT triple and more
mutants. Data was generated via qPCR human normalizer
assay and data is reported by Ct value.
Concentration Ct Ct Standard
MMLV RT Variant of RTase (nM) Mean Deviation
MMLV-II 0.625 25.520 0.047
MMLV-II L99R/E282D 0.625 24.332 0.060
MMLV-II Q68R/L99R 0.625 22.207 0.097
MMLV-II Q79R/L99R 0.625 23.789 0.012
MMLV-II Q68R/Q79R 0.625 23.629 0.038
MMLV-II Q68R/L99R/E282D 0.625 22.855 0.079
MMLV-II Q79R/L99R/E282D 0.625 23.095 0.035
MMLV-II Q68R/Q79R/E282D 0.625 22.526 0.027
MMLV-II Q68R/Q79R/L99R 0.625 22.099 0.018
MMLV-II 0.625 21.056 0.023
Q68R/Q79R/L99R/E282D
MMLV-II 0.625 21.833 0.031
Q68R/Q79R/L99K/E282D
MMLV-II 0.625 23.607 0.031
Q68R/Q79R/L99N/E282D
MMLV-II 0.625 23.858 0.029
Q68I/Q79R/L99R/E282D
MMLV-II 0.625 22.615 0.054
Q68K/Q79R/L99R/E282D
MMLV-II 0.625 28.866 0.008
Q68R/Q79H/L99R/E282D
MMLV-II 0.625 23.283 0.085
Q68R/Q79I/L99R/E282D
MMLV-II 0.625 25.073 0.097
Q68R/Q79R/L99R/E282M
MMLV-II 0.625 22.331 0.048
Q68R/Q79R/L99R/E282W
MMLV-II 0.625 23.271 0.065
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 0.625 22.133 0.018
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 0.625 23.344 0.037
Q68I/Q79H/L99K/E282M
MMLV-II 0.625 25.255 0.058
I61M/Q68I/Q79H/L99K/E282M
MMLV-II 2.5 22.154 0.052
MMLV-II L99R/E282D 2.5 21.501 0.054
MMLV-II Q68R/L99R 2.5 21.151 0.048
MMLV-II Q79R/L99R 2.5 21.229 0.163
MMLV-II Q68R/Q79R 2.5 21.228 0.054
MMLV-II Q68R/L99R/E282D 2.5 21.126 0.030
MMLV-II Q79R/L99R/E282D 2.5 21.418 0.033
MMLV-II Q68R/Q79R/E282D 2.5 21.011 0.052
MMLV-II Q68R/Q79R/L99R 2.5 20.953 0.041
MMLV-II 2.5 21.113 0.108
Q68R/Q79R/L99R/E282D
MMLV-II 2.5 20.906 0.081
Q68R/Q79R/L99K/E282D
MMLV-II 2.5 21.196 0.029
Q68R/Q79R/L99N/E282D
MMLV-II 2.5 21.369 0.009
Q68I/Q79R/L99R/E282D
MMLV-II 2.5 20.960 0.030
Q68K/Q79R/L99R/E282D
MMLV-II 2.5 26.167 0.038
Q68R/Q79H/L99R/E282D
MMLV-II 2.5 21.012 0.056
Q68R/Q79I/L99R/E282D
MMLV-II 2.5 21.277 0.036
Q68R/Q79R/L99R/E282M
MMLV-II 2.5 20.944 0.020
Q68R/Q79R/L99R/E282W
MMLV-II 2.5 21.320 0.009
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 2.5 21.095 0.013
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 2.5 21.329 0.047
Q68I/Q79H/L99K/E282M
MMLV-II 2.5 22.159 0.031
I61M/Q68I/Q79H/L99K/E282M
MMLV-II 10 21.575 0.101
MMLV-II L99R/E282D 10 21.546 0.041
MMLV-II Q68R/L99R 10 21.343 0.021
MMLV-II Q79R/L99R 10 21.387 0.016
MMLV-II Q68R/Q79R 10 21.147 0.032
MMLV-II Q68R/L99R/E282D 10 21.265 0.076
MMLV-II Q79R/L99R/E282D 10 21.250 0.036
MMLV-II Q68R/Q79R/E282D 10 21.135 0.015
MMLV-II Q68R/Q79R/L99R 10 21.051 0.036
MMLV-II 10 21.159 0.065
Q68R/Q79R/L99R/E282D
MMLV-II 10 21.056 0.032
Q68R/Q79R/L99K/E282D
MMLV-II 10 21.180 0.052
Q68R/Q79R/L99N/E282D
MMLV-II 10 21.068 0.069
Q68I/Q79R/L99R/E282D
MMLV-II 10 21.065 0.053
Q68K/Q79R/L99R/E282D
MMLV-II 10 21.683 0.075
Q68R/Q79H/L99R/E282D
MMLV-II 10 21.152 0.064
Q68R/Q79I/L99R/E282D
MMLV-II 10 21.029 0.055
Q68R/Q79R/L99R/E282M
MMLV-II 10 21.214 0.052
Q68R/Q79R/L99R/E282W
MMLV-II 10 21.391 0.051
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 10 21.307 0.038
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 10 21.583 0.019
Q68I/Q79H/L99K/E282M
MMLV-II 10 21.759 0.029
I61M/Q68I/Q79H/L99K/E282M
TABLE 14
One-Step cDNA synthesis by MMLV RT triple and more
mutants. Data was generated via qPCR human normalizer
assay and data is reported by Ct value.
Concentration Ct Ct Standard
MMLV RT Variant of RTase (nM) Mean Deviation
MMLV-II 0.625 22.153 0.122
MMLV-II L99R/E282D 0.625 21.713 0.111
MMLV-II Q68R/L99R 0.625 21.334 0.167
MMLV-II Q79R/L99R 0.625 21.398 0.069
MMLV-II Q68R/Q79R 0.625 21.546 0.096
MMLV-II Q68R/L99R/E282D 0.625 21.112 0.149
MMLV-II Q79R/L99R/E282D 0.625 21.260 0.104
MMLV-II Q68R/Q79R/E282D 0.625 21.014 0.102
MMLV-II Q68R/Q79R/L99R 0.625 20.338 0.042
MMLV-II 0.625 19.537 0.120
Q68R/Q79R/L99R/E282D
MMLV-II 0.625 20.516 0.131
Q68R/Q79R/L99K/E282D
MMLV-II 0.625 20.960 0.023
Q68R/Q79R/L99N/E282D
MMLV-II 0.625 21.325 0.088
Q68I/Q79R/L99R/E282D
MMLV-II 0.625 20.602 0.038
Q68K/Q79R/L99R/E282D
MMLV-II 0.625 23.889 0.042
Q68R/Q79H/L99R/E282D
MMLV-II 0.625 21.375 0.035
Q68R/Q79I/L99R/E282D
MMLV-II 0.625 21.805 0.054
Q68R/Q79R/L99R/E282M
MMLV-II 0.625 20.229 0.085
Q68R/Q79R/L99R/E282W
MMLV-II 0.625 20.972 0.037
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 0.625 20.225 0.042
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 0.625 20.578 0.061
Q68I/Q79H/L99K/E282M
MMLV-II 0.625 21.107 0.101
I61M/Q68I/Q79H/L99K/E282M
MMLV-II 2.5 20.874 0.042
MMLV-II L99R/E282D 2.5 19.679 0.047
MMLV-II Q68R/L99R 2.5 19.152 0.024
MMLV-II Q79R/L99R 2.5 19.202 0.091
MMLV-II Q68R/Q79R 2.5 19.506 0.010
MMLV-II Q68R/L99R/E282D 2.5 19.142 0.060
MMLV-II Q79R/L99R/E282D 2.5 19.301 0.004
MMLV-II Q68R/Q79R/E282D 2.5 19.023 0.041
MMLV-II Q68R/Q79R/L99R 2.5 18.312 0.041
MMLV-II 2.5 17.867 0.099
Q68R/Q79R/L99R/E282D
MMLV-II 2.5 18.591 0.036
Q68R/Q79R/L99K/E282D
MMLV-II 2.5 19.123 0.097
Q68R/Q79R/L99N/E282D
MMLV-II 2.5 19.553 0.076
Q68I/Q79R/L99R/E282D
MMLV-II 2.5 18.771 0.113
Q68K/Q79R/L99R/E282D
MMLV-II 2.5 21.911 0.048
Q68R/Q79H/L99R/E282D
MMLV-II 2.5 19.298 0.146
Q68R/Q79I/L99R/E282D
MMLV-II 2.5 19.621 0.027
Q68R/Q79R/L99R/E282M
MMLV-II 2.5 18.219 0.103
Q68R/Q79R/L99R/E282W
MMLV-II 2.5 18.846 0.056
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 2.5 18.500 0.042
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 2.5 18.752 0.148
Q68I/Q79H/L99K/E282M
MMLV-II 2.5 19.445 0.098
I61M/Q68I/Q79H/L99K/E282M
MMLV-II 10 18.239 0.025
MMLV-II L99R/E282D 10 17.293 0.021
MMLV-II Q68R/L99R 10 17.144 0.032
MMLV-II Q79R/L99R 10 17.324 0.016
MMLV-II Q68R/Q79R 10 17.123 0.072
MMLV-II Q68R/L99R/E282D 10 17.082 0.088
MMLV-II Q79R/L99R/E282D 10 17.353 0.068
MMLV-II Q68R/Q79R/E282D 10 17.111 0.036
MMLV-II Q68R/Q79R/L99R 10 16.562 0.101
MMLV-II 10 16.492 0.066
Q68R/Q79R/L99R/E282D
MMLV-II 10 17.027 0.054
Q68R/Q79R/L99K/E282D
MMLV-II 10 17.335 0.080
Q68R/Q79R/L99N/E282D
MMLV-II 10 17.726 0.055
Q68I/Q79R/L99R/E282D
MMLV-II 10 17.144 0.140
Q68K/Q79R/L99R/E282D
MMLV-II 10 19.772 0.064
Q68R/Q79H/L99R/E282D
MMLV-II 10 17.424 0.020
Q68R/Q79I/L99R/E282D
MMLV-II 10 17.624 0.014
Q68R/Q79R/L99R/E282M
MMLV-II 10 16.629 0.080
Q68R/Q79R/L99R/E282W
MMLV-II 10 16.903 0.022
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 10 16.803 0.028
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 10 16.894 0.056
Q6I/Q79H/L99K/E282M
MMLV-II 10 17.509 0.058
I61M/Q68I/Q79H/L99K/E282M
e. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA by Oligo-dT or Random Priming
MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Oligo-dT or random hexamer priming conditions were adjusted for the two-step reactions and RTase concentration was normalized to 31 nM. The two-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see Tables 15 and 16).
Nine of the seventeen MMLV RTase triple or more mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase stacked mutant variants, and almost all of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The nine MMLV RTase mutant variants that were found to exhibit the highest overall activity were Q79R/L99R/E282D, Q68R/Q79R/L99R, Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99K/E282D, Q68R/Q79R/L99N/E282D, Q68K/Q79R/L99R/E282D, Q68R/Q79R/L99R/E282M, M61K/Q68R/Q79R/L99R/E282D and 161M/Q68R/Q79R/L99R/E282D.
TABLE 15
Two-Step cDNA synthesis by MMLV RT triple and more mutants
by Oligo-dT priming. Data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Temperature of Ct Ct Standard
MMLV RT Variant Reaction (° C.) Mean Deviation
MMLV-II 42 25.165 0.057
MMLV-II L99R/E282D 42 25.287 0.062
MMLV-II Q68R/L99R 42 25.026 0.035
MMLV-II Q79R/L99R 42 24.932 0.032
MMLV-II Q68R/Q79R 42 25.002 0.076
MMLV-II Q68R/L99R/E282D 42 24.964 0.068
MMLV-II Q79R/L99R/E282D 42 24.822 0.106
MMLV-II Q68R/Q79R/E282D 42 24.905 0.134
MMLV-II Q68R/Q79R/L99R 42 24.673 0.131
MMLV-II 42 24.523 0.111
Q68R/Q79R/L99R/E282D
MMLV-II 42 24.677 0.076
Q68R/Q79R/L99K/E282D
MMLV-II 42 24.635 0.087
Q68R/Q79R/L99N/E282D
MMLV-II 42 25.010 0.074
Q68I/Q79R/L99R/E282D
MMLV-II 42 24.676 0.066
Q68K/Q79R/L99R/E282D
MMLV-II 42 28.929 0.021
Q68R/Q79H/L99R/E282D
MMLV-II 42 24.932 0.039
Q68R/Q79I/L99R/E282D
MMLV-II 42 24.900 0.113
Q68R/Q79R/L99R/E282M
MMLV-II 42 24.967 0.091
Q68R/Q79R/L99R/E282W
MMLV-II 42 24.597 0.076
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 42 24.833 0.007
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 42 25.440 0.048
Q68I/Q79H/L99K/E282M
MMLV-II 42 25.679 0.050
I61M/Q68I/Q79H/L99K/E282M
MMLV-II 55 34.223 0.406
MMLV-II L99R/E282D 55 34.732 3.729
MMLV-II Q68R/L99R 55 31.509 0.169
MMLV-II Q79R/L99R 55 31.831 0.019
MMLV-II Q68R/Q79R 55 32.633 1.094
MMLV-II Q68R/L99R/E282D 55 32.089 0.075
MMLV-II Q79R/L99R/E282D 55 32.134 0.081
MMLV-II Q68R/Q79R/E282D 55 34.639 3.791
MMLV-II Q68R/Q79R/L99R 55 29.559 0.029
MMLV-II 55 28.013 0.136
Q68R/Q79R/L99R/E282D
MMLV-II 55 29.712 0.090
Q68R/Q79R/L99K/E282D
MMLV-II 55 30.442 0.224
Q68R/Q79R/L99N/E282D
MMLV-II 55 32.857 0.378
Q68I/Q79R/L99R/E282D
MMLV-II 55 31.186 0.630
Q68K/Q79R/L99R/E282D
MMLV-II 55 37.338 1.882
Q68R/Q79H/L99R/E282D
MMLV-II 55 31.830 0.120
Q68R/Q79I/L99R/E282D
MMLV-II 55 31.682 0.181
Q68R/Q79R/L99R/E282M
MMLV-II 55 32.256 0.228
Q68R/Q79R/L99R/E282W
MMLV-II 55 30.362 0.129
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 55 31.473 0.070
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 55 32.892 0.286
Q68I/Q79H/L99K/E282M
MMLV-II 55 33.872 0.131
I61M/Q68I/Q79H/L99K/E282M
TABLE 16
Two-Step cDNA synthesis by MMLV RT triple and more mutants
by random hexamer priming. Data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Temperature of Ct Ct Standard
MMLV RT Variant Reaction (° C.) Mean Deviation
MMLV-II 42 24.675 0.054
MMLV-II L99R/E282D 42 24.864 0.043
MMLV-II Q68R/L99R 42 24.577 0.066
MMLV-II Q79R/L99R 42 24.630 0.103
MMLV-II Q68R/Q79R 42 24.496 0.050
MMLV-II Q68R/L99R/E282D 42 24.549 0.059
MMLV-II Q79R/L99R/E282D 42 24.625 0.013
MMLV-II Q68R/Q79R/E282D 42 24.623 0.083
MMLV-II Q68R/Q79R/L99R 42 24.494 0.070
MMLV-II 42 24.422 0.035
Q68R/Q79R/L99R/E282D
MMLV-II 42 24.517 0.066
Q68R/Q79R/L99K/E282D
MMLV-II 42 24.324 0.059
Q68R/Q79R/L99N/E282D
MMLV-II 42 24.488 0.070
Q68I/Q79R/L99R/E282D
MMLV-II 42 24.501 0.041
Q68K/Q79R/L99R/E282D
MMLV-II 42 26.574 0.029
Q68R/Q79H/L99R/E282D
MMLV-II 42 24.496 0.055
Q68R/Q79I/L99R/E282D
MMLV-II 42 24.382 0.043
Q68R/Q79R/L99R/E282M
MMLV-II 42 24.617 0.109
Q68R/Q79R/L99R/E282W
MMLV-II 42 24.391 0.045
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 42 24.426 0.028
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 42 24.660 0.027
Q68I/Q79H/L99K/E282M
MMLV-II 42 24.949 0.052
I61M/Q68I/Q79H/L99K/E282M
MMLV-II 55 32.082 0.095
MMLV-II L99R/E282D 55 31.612 0.190
MMLV-II Q68R/L99R 55 30.349 0.041
MMLV-II Q79R/L99R 55 30.494 0.094
MMLV-II Q68R/Q79R 55 29.735 0.153
MMLV-II Q68R/L99R/E282D 55 30.724 0.045
MMLV-II Q79R/L99R/E282D 55 30.774 0.152
MMLV-II Q68R/Q79R/E282D 55 30.232 0.079
MMLV-II Q68R/Q79R/L99R 55 28.270 0.340
MMLV-II 55 26.673 0.143
Q68R/Q79R/L99R/E282D
MMLV-II 55 28.258 0.018
Q68R/Q79R/L99K/E282D
MMLV-II 55 28.973 0.116
Q68R/Q79R/L99N/E282D
MMLV-II 55 31.617 0.071
Q68I/Q79R/L99R/E282D
MMLV-II 55 28.994 0.110
Q68K/Q79R/L99R/E282D
MMLV-II 55 35.664 0.695
Q68R/Q79H/L99R/E282D
MMLV-II 55 30.265 0.116
Q68R/Q79I/L99R/E282D
MMLV-II 55 29.765 0.059
Q68R/Q79R/L99R/E282M
MMLV-II 55 30.535 0.424
Q68R/Q79R/L99R/E282W
MMLV-II 55 28.878 0.038
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 55 29.778 0.081
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 55 31.836 0.222
Q68I/Q79H/L99K/E282M
MMLV-II 55 31.984 0.223
I61M/Q68I/Q79H/L99K/E282M
f. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA Over a Wide Range of Temperatures
MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Oligo-dT or random hexamer priming conditions and reaction temperatures were adjusted for the two-step reactions and RTase concentration was normalized to 31 nM. The two-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see Tables 17 and 18).
Six of the nine MMLV RTase triple or more mutant variants were found to exhibit high overall activity as compared to the other MMLV RTase stacked mutant variants over a wide range of temperatures, spanning from 37.0 to 65° C., regardless of which priming method used. All of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The six MMLV RTase mutant variants that were found to exhibit the highest overall activity at a wide range of temperatures were Q68R/Q79R/L99R, Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99K/E282D, Q68R/Q79R/L99N/E282D, 161K/Q68R/Q79R/L99R/E282D and 161 M/Q68R/Q79R/L99R/E282D.
TABLE 17
Two-Step cDNA synthesis by MMLV RT triple and more mutants
by Oligo-dT priming. Data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Temperature of Ct Ct Standard
MMLV RT Variant Reaction (° C.) Mean Deviation
MMLV-II 37.0 26.593 0.020
MMLV-II Q79R/L99R/E282D 37.0 25.713 0.024
MMLV-II Q68R/Q79R/L99R 37.0 25.164 0.059
MMLV-II 37.0 25.163 0.035
Q68R/Q79R/L99R/E282D
MMLV-II 37.0 25.135 0.078
Q68R/Q79R/L99K/E282D
MMLV-II 37.0 25.693 0.048
Q68R/Q79R/L99N/E282D
MMLV-II 37.0 25.491 0.062
Q68K/Q79R/L99R/E282D
MMLV-II 37.0 25.450 0.083
Q68R/Q79R/L99R/E282M
MMLV-II 37.0 25.094 0.071
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 37.0 25.356 0.034
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 37.8 26.623 0.062
MMLV-II Q79R/L99R/E282D 37.8 25.516 0.078
MMLV-II Q68R/Q79R/L99R 37.8 25.251 0.094
MMLV-II 37.8 24.987 0.050
Q68R/Q79R/L99R/E282D
MMLV-II 37.8 25.093 0.084
Q68R/Q79R/L99K/E282D
MMLV-II 37.8 25.273 0.095
Q68R/Q79R/L99N/E282D
MMLV-II 37.8 25.310 0.079
Q68K/Q79R/L99R/E282D
MMLV-II 37.8 25.545 0.044
Q68R/Q79R/L99R/E282M
MMLV-II 37.8 25.144 0.196
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 37.8 25.302 0.035
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 39.5 26.430 0.074
MMLV-II Q79R/L99R/E282D 39.5 25.067 0.026
MMLV-II Q68R/Q79R/L99R 39.5 25.138 0.050
MMLV-II 39.5 24.788 0.022
Q68R/Q79R/L99R/E282D
MMLV-II 39.5 24.842 0.071
Q68R/Q79R/L99K/E282D
MMLV-II 39.5 24.892 0.042
Q68R/Q79R/L99N/E282D
MMLV-II 39.5 25.047 0.038
Q68K/Q79R/L99R/E282D
MMLV-II 39.5 25.249 0.081
Q68R/Q79R/L99R/E282M
MMLV-II 39.5 24.845 0.130
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 39.5 25.130 0.072
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 42.0 25.485 0.052
MMLV-II Q79R/L99R/E282D 42.0 24.941 0.024
MMLV-II Q68R/Q79R/L99R 42.0 24.848 0.101
MMLV-II 42.0 24.802 0.009
Q68R/Q79R/L99R/E282D
MMLV-II 42.0 24.805 0.008
Q68R/Q79R/L99K/E282D
MMLV-II 42.0 24.744 0.076
Q68R/Q79R/L99N/E282D
MMLV-II 42.0 24.893 0.073
Q68K/Q79R/L99R/E282D
MMLV-II 42.0 24.968 0.031
Q68R/Q79R/L99R/E282M
MMLV-II 42.0 24.933 0.088
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 42.0 24.821 0.045
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 45.2 25.776 0.028
MMLV-II Q79R/L99R/E282D 45.2 24.902 0.034
MMLV-II Q68R/Q79R/L99R 45.2 24.792 0.055
MMLV-II 45.2 24.705 0.092
Q68R/Q79R/L99R/E282D
MMLV-II 45.2 24.791 0.009
Q68R/Q79R/L99K/E282D
MMLV-II 45.2 24.890 0.071
Q68R/Q79R/L99N/E282D
MMLV-II 45.2 25.420 0.101
Q68K/Q79R/L99R/E282D
MMLV-II 45.2 25.196 0.086
Q68R/Q79R/L99R/E282M
MMLV-II 45.2 24.823 0.079
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 45.2 24.720 0.006
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 47.8 27.932 0.049
MMLV-II Q79R/L99R/E282D 47.8 24.858 0.063
MMLV-II Q68R/Q79R/L99R 47.8 24.685 0.095
MMLV-II 47.8 24.689 0.067
Q68R/Q79R/L99R/E282D
MMLV-II 47.8 24.620 0.072
Q68R/Q79R/L99K/E282D
MMLV-II 47.8 24.780 0.039
Q68R/Q79R/L99N/E282D
MMLV-II 47.8 24.855 0.018
Q68K/Q79R/L99R/E282D
MMLV-II 47.8 24.961 0.040
Q68R/Q79R/L99R/E282M
MMLV-II 47.8 24.681 0.076
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 47.8 24.759 0.055
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 49.2 30.393 0.118
MMLV-II Q79R/L99R/E282D 49.2 24.974 0.090
MMLV-II Q68R/Q79R/L99R 49.2 24.794 0.056
MMLV-II 49.2 24.720 0.100
Q68R/Q79R/L99R/E282D
MMLV-II 49.2 25.007 0.096
Q68R/Q79R/L99K/E282D
MMLV-II 49.2 25.304 0.147
Q68R/Q79R/L99N/E282D
MMLV-II 49.2 25.273 0.066
Q68K/Q79R/L99R/E282D
MMLV-II 49.2 25.560 0.019
Q68R/Q79R/L99R/E282M
MMLV-II 49.2 24.719 0.177
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 49.2 25.123 0.034
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 50.0 30.870 0.210
MMLV-II Q79R/L99R/E282D 50.0 26.677 0.090
MMLV-II Q68R/Q79R/L99R 50.0 25.381 0.049
MMLV-II 50.0 24.820 0.064
Q68R/Q79R/L99R/E282D
MMLV-II 50.0 25.348 0.098
Q68R/Q79R/L99K/E282D
MMLV-II 50.0 25.287 0.064
Q68R/Q79R/L99N/E282D
MMLV-II 50.0 25.208 0.085
Q68K/Q79R/L99R/E282D
MMLV-II 50.0 25.790 0.051
Q68R/Q79R/L99R/E282M
MMLV-II 50.0 24.840 0.071
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 50.0 25.317 0.042
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 51.0 27.914 0.002
MMLV-II Q79R/L99R/E282D 51.0 25.561 0.069
MMLV-II Q68R/Q79R/L99R 51.0 25.225 0.069
MMLV-II 51.0 24.726 0.034
Q68R/Q79R/L99R/E282D
MMLV-II 51.0 25.324 0.071
Q68R/Q79R/L99K/E282D
MMLV-II 51.0 25.157 0.062
Q68R/Q79R/L99N/E282D
MMLV-II 51.0 25.275 0.039
Q68K/Q79R/L99R/E282D
MMLV-II 51.0 25.938 0.095
Q68R/Q79R/L99R/E282M
MMLV-II 51.0 25.821 0.072
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 51.0 25.053 0.044
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 51.9 28.602 0.059
MMLV-II Q79R/L99R/E282D 51.9 25.975 0.024
MMLV-II Q68R/Q79R/L99R 51.9 25.256 0.075
MMLV-II 51.9 24.903 0.050
Q68R/Q79R/L99R/E282D
MMLV-II 51.9 25.163 0.169
Q68R/Q79R/L99K/E282D
MMLV-II 51.9 25.272 0.011
Q68R/Q79R/L99N/E282D
MMLV-II 51.9 25.491 0.075
Q68K/Q79R/L99R/E282D
MMLV-II 51.9 25.878 0.038
Q68R/Q79R/L99R/E282M
MMLV-II 51.9 26.071 0.044
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 51.9 25.419 0.067
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 53.8 26.412 0.082
MMLV-II Q79R/L99R/E282D 53.8 25.558 0.063
MMLV-II Q68R/Q79R/L99R 53.8 24.969 0.065
MMLV-II 53.8 25.356 0.063
Q68R/Q79R/L99R/E282D
MMLV-II 53.8 25.460 0.056
Q68R/Q79R/L99K/E282D
MMLV-II 53.8 25.769 0.118
Q68R/Q79R/L99N/E282D
MMLV-II 53.8 26.251 0.103
Q68K/Q79R/L99R/E282D
MMLV-II 53.8 26.310 0.174
Q68R/Q79R/L99R/E282M
MMLV-II 53.8 25.701 0.106
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 53.8 26.412 0.082
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 56.5 29.343 0.085
MMLV-II Q79R/L99R/E282D 56.5 26.885 0.083
MMLV-II Q68R/Q79R/L99R 56.5 25.736 0.015
MMLV-II 56.5 25.223 0.016
Q68R/Q79R/L99R/E282D
MMLV-II 56.5 25.900 0.039
Q68R/Q79R/L99K/E282D
MMLV-II 56.5 25.930 0.031
Q68R/Q79R/L99N/E282D
MMLV-II 56.5 25.869 0.204
Q68K/Q79R/L99R/E282D
MMLV-II 56.5 26.622 0.067
Q68R/Q79R/L99R/E282M
MMLV-II 56.5 25.817 0.089
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 56.5 26.290 0.009
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 59.9 29.693 0.047
MMLV-II Q79R/L99R/E282D 59.9 27.820 0.014
MMLV-II Q68R/Q79R/L99R 59.9 26.069 0.057
MMLV-II 59.9 25.374 0.061
Q68R/Q79R/L99R/E282D
MMLV-II 59.9 26.066 0.053
Q68R/Q79R/L99K/E282D
MMLV-II 59.9 25.873 0.018
Q68R/Q79R/L99N/E282D
MMLV-II 59.9 26.278 0.073
Q68K/Q79R/L99R/E282D
MMLV-II 59.9 27.068 0.075
Q68R/Q79R/L99R/E282M
MMLV-II 59.9 26.863 0.025
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 59.9 26.176 0.072
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 62.6 29.731 0.092
MMLV-II Q79R/L99R/E282D 62.6 27.161 0.035
MMLV-II Q68R/Q79R/L99R 62.6 25.929 0.026
MMLV-II 62.6 25.303 0.074
Q68R/Q79R/L99R/E282D
MMLV-II 62.6 25.907 0.003
Q68R/Q79R/L99K/E282D
MMLV-II 62.6 26.145 0.053
Q68R/Q79R/L99N/E282D
MMLV-II 62.6 26.181 0.056
Q68K/Q79R/L99R/E282D
MMLV-II 62.6 27.134 0.015
Q68R/Q79R/L99R/E282M
MMLV-II 62.6 26.025 0.178
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 62.6 26.304 0.041
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 64.2 26.809 0.080
MMLV-II Q79R/L99R/E282D 64.2 27.325 0.038
MMLV-II Q68R/Q79R/L99R 64.2 26.131 0.018
MMLV-II 64.2 25.542 0.135
Q68R/Q79R/L99R/E282D
MMLV-II 64.2 26.408 0.093
Q68R/Q79R/L99K/E282D
MMLV-II 64.2 26.734 0.040
Q68R/Q79R/L99N/E282D
MMLV-II 64.2 30.589 0.128
Q68K/Q79R/L99R/E282D
MMLV-II 64.2 26.262 0.090
Q68R/Q79R/L99R/E282M
MMLV-II 64.2 27.594 0.118
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 64.2 27.062 0.051
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 65.0 30.277 0.050
MMLV-II Q79R/L99R/E282D 65.0 27.119 0.065
MMLV-II Q68R/Q79R/L99R 65.0 26.078 0.025
MMLV-II 65.0 25.583 0.068
Q68R/Q79R/L99R/E282D
MMLV-II 65.0 25.906 0.080
Q68R/Q79R/L99K/E282D
MMLV-II 65.0 26.943 0.058
Q68R/Q79R/L99N/E282D
MMLV-II 65.0 26.413 0.067
Q68K/Q79R/L99R/E282D
MMLV-II 65.0 28.233 0.075
Q68R/Q79R/L99R/E282M
MMLV-II 65.0 25.778 0.129
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 65.0 27.345 0.015
I61M/Q68R/Q79R/L99R/E282D
TABLE 18
Two-Step cDNA synthesis by MMLV RT triple and more mutants
by random hexamer priming. Data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Temperature of Ct Ct Standard
MMLV RT Variant Reaction (° C.) Mean Deviation
MMLV-II 37.0 25.827 0.120
MMLV-II Q79R/L99R/E282D 37.0 25.616 0.094
MMLV-II Q68R/Q79R/L99R 37.0 24.747 0.041
MMLV-II 37.0 24.595 0.034
Q68R/Q79R/L99R/E282D
MMLV-II 37.0 24.917 0.078
Q68R/Q79R/L99K/E282D
MMLV-II 37.0 24.817 0.024
Q68R/Q79R/L99N/E282D
MMLV-II 37.0 24.757 0.032
Q68K/Q79R/L99R/E282D
MMLV-II 37.0 24.754 0.062
Q68R/Q79R/L99R/E282M
MMLV-II 37.0 24.883 0.106
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 37.0 24.776 0.028
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 37.8 25.609 0.038
MMLV-II Q79R/L99R/E282D 37.8 25.300 0.061
MMLV-II Q68R/Q79R/L99R 37.8 24.822 0.037
MMLV-II 37.8 24.690 0.044
Q68R/Q79R/L99R/E282D
MMLV-II 37.8 24.884 0.033
Q68R/Q79R/L99K/E282D
MMLV-II 37.8 24.665 0.022
Q68R/Q79R/L99N/E282D
MMLV-II 37.8 24.846 0.021
Q68K/Q79R/L99R/E282D
MMLV-II 37.8 24.882 0.043
Q68R/Q79R/L99R/E282M
MMLV-II 37.8 24.846 0.059
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 37.8 24.723 0.023
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 39.5 25.455 0.020
MMLV-II Q79R/L99R/E282D 39.5 24.790 0.109
MMLV-II Q68R/Q79R/L99R 39.5 24.712 0.050
MMLV-II 39.5 24.543 0.005
Q68R/Q79R/L99R/E282D
MMLV-II 39.5 24.714 0.035
Q68R/Q79R/L99K/E282D
MMLV-II 39.5 24.520 0.084
Q68R/Q79R/L99N/E282D
MMLV-II 39.5 24.752 0.047
Q68K/Q79R/L99R/E282D
MMLV-II 39.5 24.850 0.054
Q68R/Q79R/L99R/E282M
MMLV-II 39.5 24.698 0.059
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 39.5 24.682 0.024
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 42.0 25.136 0.034
MMLV-II Q79R/L99R/E282D 42.0 24.760 0.052
MMLV-II Q68R/Q79R/L99R 42.0 24.637 0.037
MMLV-II 42.0 24.449 0.008
Q68R/Q79R/L99R/E282D
MMLV-II 42.0 24.650 0.068
Q68R/Q79R/L99K/E282D
MMLV-II 42.0 24.477 0.055
Q68R/Q79R/L99N/E282D
MMLV-II 42.0 24.624 0.029
Q68K/Q79R/L99R/E282D
MMLV-II 42.0 24.627 0.044
Q68R/Q79R/L99R/E282M
MMLV-II 42.0 24.718 0.083
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 42.0 24.532 0.021
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 45.2 25.079 0.017
MMLV-II Q79R/L99R/E282D 45.2 24.624 0.026
MMLV-II Q68R/Q79R/L99R 45.2 24.525 0.021
MMLV-II 45.2 24.430 0.014
Q68R/Q79R/L99R/E282D
MMLV-II 45.2 24.525 0.037
Q68R/Q79R/L99K/E282D
MMLV-II 45.2 34.853 0.705
Q68R/Q79R/L99N/E282D
MMLV-II 45.2 24.653 0.055
Q68K/Q79R/L99R/E282D
MMLV-II 45.2 24.552 0.060
Q68R/Q79R/L99R/E282M
MMLV-II 45.2 24.595 0.027
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 45.2 24.493 0.016
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 47.8 25.346 0.007
MMLV-II Q79R/L99R/E282D 47.8 24.521 0.097
MMLV-II Q68R/Q79R/L99R 47.8 24.605 0.018
MMLV-II 47.8 24.333 0.107
Q68R/Q79R/L99R/E282D
MMLV-II 47.8 24.516 0.043
Q68R/Q79R/L99K/E282D
MMLV-II 47.8 24.527 0.026
Q68R/Q79R/L99N/E282D
MMLV-II 47.8 24.539 0.064
Q68K/Q79R/L99R/E282D
MMLV-II 47.8 24.631 0.019
Q68R/Q79R/L99R/E282M
MMLV-II 47.8 24.227 0.260
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 47.8 24.441 0.030
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 49.2 25.791 0.064
MMLV-II Q79R/L99R/E282D 49.2 24.700 0.033
MMLV-II Q68R/Q79R/L99R 49.2 24.658 0.008
MMLV-II 49.2 24.471 0.069
Q68R/Q79R/L99R/E282D
MMLV-II 49.2 24.590 0.024
Q68R/Q79R/L99K/E282D
MMLV-II 49.2 24.482 0.099
Q68R/Q79R/L99N/E282D
MMLV-II 49.2 24.549 0.028
Q68K/Q79R/L99R/E282D
MMLV-II 49.2 24.753 0.030
Q68R/Q79R/L99R/E282M
MMLV-II 49.2 24.499 0.157
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 49.2 24.559 0.033
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 50.0 26.267 0.025
MMLV-II Q79R/L99R/E282D 50.0 24.729 0.047
MMLV-II Q68R/Q79R/L99R 50.0 24.462 0.040
MMLV-II 50.0 24.412 0.035
Q68R/Q79R/L99R/E282D
MMLV-II 50.0 24.438 0.090
Q68R/Q79R/L99K/E282D
MMLV-II 50.0 24.509 0.050
Q68R/Q79R/L99N/E282D
MMLV-II 50.0 24.405 0.059
Q68K/Q79R/L99R/E282D
MMLV-II 50.0 24.547 0.041
Q68R/Q79R/L99R/E282M
MMLV-II 50.0 24.504 0.005
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 50.0 24.481 0.009
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 51.0 27.277 0.058
MMLV-II Q79R/L99R/E282D 51.0 25.694 0.104
MMLV-II Q68R/Q79R/L99R 51.0 24.579 0.037
MMLV-II 51.0 24.364 0.019
Q68R/Q79R/L99R/E282D
MMLV-II 51.0 24.849 0.041
Q68R/Q79R/L99K/E282D
MMLV-II 51.0 24.899 0.121
Q68R/Q79R/L99N/E282D
MMLV-II 51.0 24.980 0.048
Q68K/Q79R/L99R/E282D
MMLV-II 51.0 25.292 0.065
Q68R/Q79R/L99R/E282M
MMLV-II 51.0 25.147 0.100
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 51.0 25.034 0.075
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 51.9 28.797 0.055
MMLV-II Q79R/L99R/E282D 51.9 26.585 0.011
MMLV-II Q68R/Q79R/L99R 51.9 25.021 0.036
MMLV-II 51.9 24.763 0.028
Q68R/Q79R/L99R/E282D
MMLV-II 51.9 25.392 0.012
Q68R/Q79R/L99K/E282D
MMLV-II 51.9 25.543 0.087
Q68R/Q79R/L99N/E282D
MMLV-II 51.9 25.549 0.058
Q68K/Q79R/L99R/E282D
MMLV-II 51.9 26.025 0.065
Q68R/Q79R/L99R/E282M
MMLV-II 51.9 26.087 0.024
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 51.9 25.756 0.054
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 53.8 30.985 0.073
MMLV-II Q79R/L99R/E282D 53.8 29.356 0.044
MMLV-II Q68R/Q79R/L99R 53.8 26.370 0.041
MMLV-II 53.8 25.580 0.049
Q68R/Q79R/L99R/E282D
MMLV-II 53.8 26.682 0.029
Q68R/Q79R/L99K/E282D
MMLV-II 53.8 26.438 0.031
Q68R/Q79R/L99N/E282D
MMLV-II 53.8 27.024 0.042
Q68K/Q79R/L99R/E282D
MMLV-II 53.8 28.314 0.051
Q68R/Q79R/L99R/E282M
MMLV-II 53.8 27.489 0.025
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 53.8 27.871 0.118
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 56.5 33.313 0.164
MMLV-II Q79R/L99R/E282D 56.5 32.626 0.113
MMLV-II Q68R/Q79R/L99R 56.5 30.047 0.089
MMLV-II 56.5 29.183 0.155
Q68R/Q79R/L99R/E282D
MMLV-II 56.5 30.750 0.051
Q68R/Q79R/L99K/E282D
MMLV-II 56.5 30.403 0.095
Q68R/Q79R/L99N/E282D
MMLV-II 56.5 31.707 0.111
Q68K/Q79R/L99R/E282D
MMLV-II 56.5 31.878 0.093
Q68R/Q79R/L99R/E282M
MMLV-II 56.5 32.235 0.291
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 56.5 32.395 0.105
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 59.9 34.408 0.498
MMLV-II Q79R/L99R/E282D 59.9 36.798 2.131
MMLV-II Q68R/Q79R/L99R 59.9 33.997 0.035
MMLV-II 59.9 32.009 0.051
Q68R/Q79R/L99R/E282D
MMLV-II 59.9 33.685 0.317
Q68R/Q79R/L99K/E282D
MMLV-II 59.9 33.083 0.163
Q68R/Q79R/L99N/E282D
MMLV-II 59.9 34.160 0.066
Q68K/Q79R/L99R/E282D
MMLV-II 59.9 33.650 0.161
Q68R/Q79R/L99R/E282M
MMLV-II 59.9 33.341 0.096
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 59.9 34.439 0.222
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 62.6 35.163 0.447
MMLV-II Q79R/L99R/E282D 62.6 37.138 1.603
MMLV-II Q68R/Q79R/L99R 62.6 34.108 0.604
MMLV-II 62.6 32.539 0.060
Q68R/Q79R/L99R/E282D
MMLV-II 62.6 34.175 0.421
Q68R/Q79R/L99K/E282D
MMLV-II 62.6 33.726 0.622
Q68R/Q79R/L99N/E282D
MMLV-II 62.6 34.376 0.408
Q68K/Q79R/L99R/E282D
MMLV-II 62.6 33.792 0.231
Q68R/Q79R/L99R/E282M
MMLV-II 62.6 33.768 0.387
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 62.6 34.428 0.085
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 64.2 37.284 0.764
MMLV-II Q79R/L99R/E282D 64.2 36.661 0.192
MMLV-II Q68R/Q79R/L99R 64.2 34.463 0.213
MMLV-II 64.2 32.992 0.023
Q68R/Q79R/L99R/E282D
MMLV-II 64.2 34.805 0.472
Q68R/Q79R/L99K/E282D
MMLV-II 64.2 34.060 0.043
Q68R/Q79R/L99N/E282D
MMLV-II 64.2 34.508 0.302
Q68K/Q79R/L99R/E282D
MMLV-II 64.2 34.481 0.078
Q68R/Q79R/L99R/E282M
MMLV-II 64.2 34.231 0.253
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 64.2 35.049 0.885
I61M/Q68R/Q79R/L99R/E282D
MMLV-II 65.0 35.809 0.511
MMLV-II Q79R/L99R/E282D 65.0 35.932 0.372
MMLV-II Q68R/Q79R/L99R 65.0 34.979 0.856
MMLV-II 65.0 33.293 0.319
Q68R/Q79R/L99R/E282D
MMLV-II 65.0 34.974 0.536
Q68R/Q79R/L99K/E282D
MMLV-II 65.0 34.862 0.268
Q68R/Q79R/L99N/E282D
MMLV-II 65.0 34.363 0.201
Q68K/Q79R/L99R/E282D
MMLV-II 65.0 34.687 0.666
Q68R/Q79R/L99R/E282M
MMLV-II 65.0 34.246 0.563
I61K/Q68R/Q79R/L99R/E282D
MMLV-II 65.0 34.872 0.467
I61M/Q68R/Q79R/L99R/E282D
Example 6: Reverse Transcriptase Mutant Evaluation by Oligo dT or Random Priming This example demonstrates the procedure used to evaluate each mutant RTase's ability to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) compared to the base construct of MMLV RTase. The mutant MMLV RTases were tested by two priming conditions: Oligo dT only and random hexamer priming using a standard two-step cDNA synthesis as described in Example 5.
The reactions were analyzed and reported by Ct value (Tables 19 and 20). Four mutant variants of MMLV RTase showed an increase in the overall activity using oligo dT priming compared to the base construct, Q299E, T332E and V433R. Eight mutant variants of MMLV RTase showed an increase in the overall activity using random priming compared to the base construct, P76R, L82R, I125R, Y271A, L280A, L280R, T328R and V433R.
TABLE 19
Two-Step cDNA Synthesis by MMLV-RT single mutants using
oligo dT priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 40.000 0.000
MMLV-II D209A 40.000 0.000
MMLV-II D209E 40.000 0.000
MMLV-II D209R 40.000 0.000
MMLV-II D83 A 40.000 0.000
MMLV-II D83E 40.000 0.000
MMLV-II D83R 40.000 0.000
MMLV-II E201A 40.000 0.000
MMLV-II E201D 40.000 0.000
MMLV-II E201R 40.000 0.000
MMLV-II E367A 40.000 0.000
MMLV-II E367D 40.000 0.000
MMLV-II E367R 40.000 0.000
MMLV-II E596A 40.000 0.000
MMLV-II E596D 40.000 0.000
MMLV-II E596R 40.000 0.000
MMLV-II F210A 40.000 0.000
MMLV-II F210E 40.000 0.000
MMLV-II F210R 40.000 0.000
MMLV-II F369A 40.000 0.000
MMLV-II F369E 40.000 0.000
MMLV-II F369R 40.000 0.000
MMLV-II G308A 40.000 0.000
MMLV-II G308E 40.000 0.000
MMLV-II G308R 40.000 0.000
MMLV-II G331A 40.000 0.000
MMLV-II G331E 40.000 0.000
MMLV-II G331R 40.000 0.000
MMLV-II G73A 40.000 0.000
MMLV-II G73E 40.000 0.000
MMLV-II G73R 40.000 0.000
MMLV-II H77A 40.000 0.000
MMLV-II H77E 40.000 0.000
MMLV-II H77R 40.000 0.000
MMLV-II I125A 40.000 0.000
MMLV-II I125E 40.000 0.000
MMLV-II I125R 40.000 0.000
MMLV-II I212A 40.000 0.000
MMLV-II I212E 40.000 0.000
MMLV-II I212R 40.000 0.000
MMLV-II I593A 40.000 0.000
MMLV-II I593E 40.000 0.000
MMLV-II I593R 40.000 0.000
MMLV-II I597A 40.000 0.000
MMLV-II I597E 40.000 0.000
MMLV-II I597R 40.000 0.000
MMLV-II K285A 40.000 0.000
MMLV-II K285E 40.000 0.000
MMLV-II K285R 40.000 0.000
MMLV-II K348A 40.000 0.000
MMLV-II K348E 40.000 0.000
MMLV-II K348R 40.000 0.000
MMLV-II L198A 40.000 0.000
MMLV-II L198E 40.000 0.000
MMLV-II L198R 40.000 0.000
MMLV-II L280A 40.000 0.000
MMLV-II L280E 40.000 0.000
MMLV-II L280R 40.000 0.000
MMLV-II L352A 40.000 0.000
MMLV-II L352E 40.000 0.000
MMLV-II L352R 40.000 0.000
MMLV-II L357A 40.000 0.000
MMLV-II L357E 40.000 0.000
MMLV-II L357R 40.000 0.000
MMLV-II L82A 40.000 0.000
MMLV-II L82E 40.000 0.000
MMLV-II L82R 40.000 0.000
MMLV-II N335A 39.787 0.302
MMLV-II N335E 40.000 0.000
MMLV-II N335R 40.000 0.000
MMLV-II P76A 40.000 0.000
MMLV-II P76E 40.000 0.000
MMLV-II P76R 40.000 0.000
MMLV-II Q213A 40.000 0.000
MMLV-II Q213E 40.000 0.000
MMLV-II Q213R 40.000 0.000
MMLV-II Q299A 40.000 0.000
MMLV-II Q299E 37.177 3.993
MMLV-II Q299R 40.000 0.000
MMLV-II Q654A 40.000 0.000
MMLV-II Q654E 40.000 0.000
MMLV-II Q654R 40.000 0.000
MMLV-II R205A 40.000 0.000
MMLV-II R205E 39.947 0.075
MMLV-II R205K 40.000 0.000
MMLV-II R211A 40.000 0.000
MMLV-II R211E 40.000 0.000
MMLV-II R211K 40.000 0.000
MMLV-II R311A 40.000 0.000
MMLV-II R311E 40.000 0.000
MMLV-II R311K 40.000 0.000
MMLV-II R389A 40.000 0.000
MMLV-II R389E 40.000 0.000
MMLV-II R389K 40.000 0.000
MMLV-II R650A 40.000 0.000
MMLV-II R650E 40.000 0.000
MMLV-II R650K 40.000 0.000
MMLV-II R657A 40.000 0.000
MMLV-II R657E 39.965 0.050
MMLV-II R657K 40.000 0.000
MMLV-II S67A 40.000 0.000
MMLV-II S67E 40.000 0.000
MMLV-II S67R 36.816 0.703
MMLV-II T328A 40.000 0.000
MMLV-II T328E 40.000 0.000
MMLV-II T328R 40.000 0.000
MMLV-II T332A 39.750 0.354
MMLV-II T332E 38.461 2.177
MMLV-II T332R 40.000 0.000
MMLV-II V129A 40.000 0.000
MMLV-II V129E 40.000 0.000
MMLV-II V129R 40.000 0.000
MMLV-II V433A 40.000 0.000
MMLV-II V433E 40.000 0.000
MMLV-II V433R 38.884 0.806
MMLV-II V476A 40.000 0.000
MMLV-II V476E 40.000 0.000
MMLV-II V476R 40.000 0.000
MMLV-II Y271A 40.000 0.000
MMLV-II Y271E 40.000 0.000
MMLV-II Y271R 40.000 0.000
MMLV-IV 31.467 0.190
TABLE 20
Two-Step cDNA Synthesis by MMLV-RT single mutants using
random priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 40.000 0.000
MMLV-II D209A 40.000 0.000
MMLV-II D209E 40.000 0.000
MMLV-II D209R 40.000 0.000
MMLV-II D83A 40.000 0.000
MMLV-II D83E 40.000 0.000
MMLV-II D83R 40.000 0.000
MMLV-II E201A 40.000 0.000
MMLV-II E201D 40.000 0.000
MMLV-II E201R 40.000 0.000
MMLV-II E367A 40.000 0.000
MMLV-II E367D 40.000 0.000
MMLV-II E367R 40.000 0.000
MMLV-II E596A 40.000 0.000
MMLV-II E596D 40.000 0.000
MMLV-II E596R 40.000 0.000
MMLV-II F210A 40.000 0.000
MMLV-II F210E 40.000 0.000
MMLV-II F210R 40.000 0.000
MMLV-II F369A 40.000 0.000
MMLV-II F369E 40.000 0.000
MMLV-II F369R 40.000 0.000
MMLV-II G308A 40.000 0.000
MMLV-II G308E 40.000 0.000
MMLV-II G308R 40.000 0.000
MMLV-II G331A 40.000 0.000
MMLV-II G331E 40.000 0.000
MMLV-II G331R 40.000 0.000
MMLV-II G73A 40.000 0.000
MMLV-II G73E 40.000 0.000
MMLV-II G73R 40.000 0.000
MMLV-II H77A 39.708 0.412
MMLV-II H77E 40.000 0.000
MMLV-II H77R 40.000 0.000
MMLV-II I125A 40.000 0.000
MMLV-II I125E 40.000 0.000
MMLV-II I125R 39.449 0.779
MMLV-II I212A 40.000 0.000
MMLV-II I212E 40.000 0.000
MMLV-II I212R 40.000 0.000
MMLV-II I593A 40.000 0.000
MMLV-II I593E 40.000 0.000
MMLV-II I593R 40.000 0.000
MMLV-II I597A 40.000 0.000
MMLV-II I597E 40.000 0.000
MMLV-II I597R 40.000 0.000
MMLV-II K285A 40.000 0.000
MMLV-II K285E 40.000 0.000
MMLV-II K285R 39.783 0.308
MMLV-II K348A 40.000 0.000
MMLV-II K348E 40.000 0.000
MMLV-II K348R 40.000 0.000
MMLV-II L198A 40.000 0.000
MMLV-II L198E 40.000 0.000
MMLV-II L198R 40.000 0.000
MMLV-II L280A 39.503 0.703
MMLV-II L280E 40.000 0.000
MMLV-II L280R 38.762 1.751
MMLV-II L352A 39.778 0.313
MMLV-II L352E 40.000 0.000
MMLV-II L352R 40.000 0.000
MMLV-II L357A 40.000 0.000
MMLV-II L357E 40.000 0.000
MMLV-II L357R 40.000 0.000
MMLV-II L82A 40.000 0.000
MMLV-II L82E 39.673 0.462
MMLV-II L82R 38.926 1.518
MMLV-II N335A 39.876 0.175
MMLV-II N335E 40.000 0.000
MMLV-II N335R 39.861 0.196
MMLV-II P76A 40.000 0.000
MMLV-II P76E 40.000 0.000
MMLV-II P76R 39.535 0.658
MMLV-II Q213A 40.000 0.000
MMLV-II Q213E 40.000 0.000
MMLV-II Q213R 40.000 0.000
MMLV-II Q299A 40.000 0.000
MMLV-II Q299E 40.000 0.000
MMLV-II Q299R 40.000 0.000
MMLV-II Q654A 40.000 0.000
MMLV-II Q654E 40.000 0.000
MMLV-II Q654R 40.000 0.000
MMLV-II R205A 39.811 0.267
MMLV-II R205E 40.000 0.000
MMLV-II R205K 40.000 0.000
MMLV-II R211A 40.000 0.000
MMLV-II R211E 40.000 0.000
MMLV-II R211K 40.000 0.000
MMLV-II R311A 40.000 0.000
MMLV-II R311E 40.000 0.000
MMLV-II R311K 40.000 0.000
MMLV-II R389A 40.000 0.000
MMLV-II R389E 40.000 0.000
MMLV-II R389K 40.000 0.000
MMLV-II R650A 40.000 0.000
MMLV-II R650E 40.000 0.000
MMLV-II R650K 40.000 0.000
MMLV-II R657A 40.000 0.000
MMLV-II R657E 40.000 0.000
MMLV-II R657K 40.000 0.000
MMLV-II S67A 40.000 0.000
MMLV-II S67E 39.435 0.800
MMLV-II S67R 38.209 0.977
MMLV-II T328A 40.000 0.000
MMLV-II T328E 40.000 0.000
MMLV-II T328R 39.478 0.739
MMLV-II T332A 40.000 0.000
MMLV-II T332E 40.000 0.000
MMLV-II T332R 40.000 0.000
MMLV-II V129A 40.000 0.000
MMLV-II V129E 40.000 0.000
MMLV-II V129R 40.000 0.000
MMLV-II V433A 40.000 0.000
MMLV-II V433E 40.000 0.000
MMLV-II V433R 38.071 1.452
MMLV-II V476A 40.000 0.000
MMLV-II V476E 40.000 0.000
MMLV-II V476R 40.000 0.000
MMLV-II Y271A 39.466 0.755
MMLV-II Y271E 40.000 0.000
MMLV-II Y271R 40.000 0.000
MMLV-IV 31.850 0.183
In addition to the increased activity demonstrated in the MMLV RTase mutations Q299E, T332E, and V433R (Table 19), and the MMLV RTase mutations P76R, L82R, I125R, Y271A, L280A, L280R, T328R, and V433R (Table 20), further MMLV RTase mutations were selected by rational design and introduced by site-directed mutagenesis using standard PCR conditions and primers (Table 21).
TABLE 21
Sequences of primers used for cloning of MMLV RTase
base construct and mutants into pET28b. All primers were
ordered as DNA oligos from Integrated DNA Technologies.
SEQ ID
NO: Primer Name Primer Sequence (5′-3′)
700 MMLV V433R AGTTGACGATGGGTCAACCCTTACGTATCTTGGCTCCA
SDM F CATGCTGTAGA
701 MMLV V433R TCTACAGCATGTGGAGCCAAGATACGTAAGGGTTGAC
SDM R CCATCGTCAACT
702 MMLV I593E CGTTATGCTTTTGCAACAGCGCATGAGCATGGCGAAA
SDM F TTTACCGCCGC
703 MMLV I593E GCGGCGGTAAATTTCGCCATGCTCATGCGCTGTTGCAA
SDM R AAGCATAACG
704 MMLV Q299E TACGCCTAAGACGCCACGCGAGTTGCGTGAATTTTTG
SDM F GGCACAGC
705 MMLV Q299E GCTGTGCCCAAAAATTCACGCAACTCGCGTGGCGTCTT
SDM R AGGCGTA
706 MMLV L82Y GATTAAGCCACATATTCAGCGCTTGTATGACCAGGGG
SDM F ATCTTGGTCC
707 MMLV L82Y GGACCAAGATCCCCTGGTCATACAAGCGCTGAATATG
SDM R TGGCTTAATC
708 MMLV L280I TGCTGAAAGAAGGTCAACGTTGGATCACTGAAGCGCG
SDM F TAAGGAGACC
709 MMLV L280I GGTCTCCTTACGCGCTTCAGTGATCCAACGTTGACCTT
SDM R CTTTCAGCA
710 MMLV V433N AGTTGACGATGGGTCAACCCTTAAACATCTTGGCTCCA
SDM F CATGCTGTAGA
711 MMLV V433N TCTACAGCATGTGGAGCCAAGATGTTTAAGGGTTGAC
SDM R CCATCGTCAACT
712 MMLV I593W CGTTATGCTTTTGCAACAGCGCATTGGCATGGCGAAAT
SDM F TTACCGCCGC
713 MMLV I593W GCGGCGGTAAATTTCGCCATGCCAATGCGCTGTTGCA
SDM R AAAGCATAACG
714 MMLV T306K GCCAGTTGCGTGAATTTTTGGGCAAAGCGGGATTCTGT
TOP CGTTTATGGATTCC
715 MMLV T306K GGAATCCATAAACGACAGAATCCCGCTTTGCCCAAAA
BTM ATTCACGCAACTGGC
The resulting plasmids were transformed into E. coli BL21(DE3) cells for protein expression and proteins isolated through affinity and ion exchange chromatography (Table 22).
TABLE 22
Sequences of MMLV RTase base construct and mutant MMLV RTase
constructs.
SEQ ID NO: Construct Construct Sequence (DNA: 5′-3′ or AA)
716 MMLV-II RTase ATGACTTTAAATATTGAGGATGAGCATCGTTTA
CATGAGACATCAAAAGAACCCGACGTGAGCTTA
GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCAAGAGGCCCGCCTGGGGATTAAGCCA
CATATTCAGCGCTTGCTGGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CTGCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGCTGACTGAAGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCCAGTTGCGTGAATTTTTGGGCACAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTAGTAATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
AACTCACGTTATGCTTTTGCAACAGCGCATATC
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTTTAA
717 MMLV-II RTase MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
MSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT
PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLVILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHI
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
718 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA
Q68R/Q79R/L99R/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA
E282D/Q299E/V433N/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
I593W TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA
CATATTCGTCGCTTGCTGGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGCTGACTGATGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCGAATTGCGTGAATTTTTGGGCACAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTACGTATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
AACTCACGTTATGCTTTTGCAACAGCGCATTGG
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTT
719 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
Q68R/Q79R/L99R/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
E282D/Q299E/V433N/ MSREARLGIKPHIRRLLDQGILVPCQSPWNTPL
I593W RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWLTDARKETVMGQPTPKI
PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
720 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA
Q68R/Q79R/L99R/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA
L2801/E282D/Q299E/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
V433N/I593W TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA
CATATTCGTCGCTTGCTGGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGATTACTGATGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCGAATTGCGTGAATTTTTGGGCACAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTACGTATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
ACTCACGTTATGCTTTTGCAACAGCGCATTGG
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTT
721 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
Q68R/Q79R/L99R/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
L280I/E282D/Q299E/ MSREARLGIKPHIRRLLDQGILVPCQSPWNTPL
V433N/I593W RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT
PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
722 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA
Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA
L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
Q299E/V433N/I593W TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA
CATATTCGTCGCTTGTATGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGATTACTGATGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCGAATTGCGTGAATTTTTGGGCACAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTACGTATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
AACTCACGTTATGCTTTTGCAACAGCGCATTGG
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTT
723 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL
Q299E/V433N/I593W RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT
PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
724 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA
Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA
L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
Q299E/T306K/V433N/ TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
I593W CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA
CATATTCGTCGCTTGTATGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGATTACTGATGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCGAATTGCGTGAATTTTTGGGCAAAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTAAACATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
AACTCACGTTATGCTTTTGCAACAGCGCATTGG
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTT
725 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL
Q299E/T306K/V433N/ RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
I593W PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT
PRELREFLGKAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLNILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHW
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
726 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA
Q68R/Q79R/L99R/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA
E282D/Q299E/T306K/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
V433R/I593E TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA
CATATTCGTCGCTTGCTGGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGCTGACTGATGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCGAATTGCGTGAATTTTTGGGCAAAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTACGTATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
AACTCACGTTATGCTTTTGCAACAGCGCATGAA
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTT
727 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
Q68R/Q79R/L99R/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
E282D/Q299E/T306K/ MSREARLGIKPHIRRLLDQGILVPCQSPWNTPL
V433R/I593E RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWLTDARKETVMGQPTPKI
PRELREFLGKAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHE
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
728 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA
Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA
L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
Q299E/V433R/I593E TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA
CATATTCGTCGCTTGTATGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGATTACTGATGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCGAATTGCGTGAATTTTTGGGCACAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTACGTATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
AACTCACGTTATGCTTTTGCAACAGCGCATGAA
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTT
729 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL
Q299E/V433R/I593E RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT
PRELREFLGTAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHE
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
730 MMLV-II ATGACTTTAAATATTGAGGATGAGCATCGTTTA
Q68R/Q79R/L82Y/ CATGAGACATCAAAAGAACCCGACGTGAGCTTA
L99R/L280I/E282D/ GGGTCAACGTGGCTTTCTGACTTCCCCCAGGCG
Q299E/T306K/V433R/ TGGGCGGAGACTGGCGGAATGGGGTTAGCTGTC
I593E CGCCAAGCACCGTTGATCATCCCGTTAAAGGCA
ACGTCTACACCTGTCTCTATCAAACAGTACCCC
ATGAGTCGTGAGGCCCGCCTGGGGATTAAGCCA
CATATTCGTCGCTTGTATGACCAGGGGATCTTG
GTCCCATGTCAATCTCCGTGGAACACCCCCCTT
CGTCCCGTGAAAAAGCCAGGTACAAACGATTAT
CGTCCAGTTCAAGATCTTCGCGAGGTCAACAAA
CGCGTAGAAGACATCCATCCGACTGTACCTAAT
CCTTATAATCTGTTATCAGGCCTGCCCCCATCG
CACCAATGGTATACAGTATTAGACTTGAAAGAC
GCGTTCTTTTGCCTGCGTCTGCACCCAACGTCT
CAGCCGCTGTTTGCGTTCGAATGGCGTGATCCT
GAAATGGGAATTTCGGGTCAGTTAACCTGGACT
CGTCTGCCCCAGGGCTTTAAAAACAGCCCCACA
TTGTTCGATGAAGCACTTCACCGTGACTTAGCA
GACTTCCGTATCCAACACCCAGACTTAATTCTG
TTACAGTATGTTGACGACCTTTTGTTGGCGGCA
ACGTCTGAACTTGACTGTCAGCAAGGCACACGC
GCGTTATTACAAACGTTAGGTAACTTAGGATAT
CGTGCGTCCGCGAAAAAGGCGCAAATTTGTCAA
AAACAGGTAAAGTACCTTGGGTATTTGCTGAAA
GAAGGTCAACGTTGGATTACTGATGCGCGTAAG
GAGACCGTAATGGGGCAGCCTACGCCTAAGACG
CCACGCGAATTGCGTGAATTTTTGGGCAAAGCG
GGATTCTGTCGTTTATGGATTCCTGGGTTCGCT
GAAATGGCTGCACCCCTGTACCCCTTAACAAAA
ACAGGGACGCTTTTCAACTGGGGGCCAGACCAG
CAAAAGGCGTATCAGGAGATCAAACAAGCTTTG
TTGACCGCACCCGCGTTGGGTCTTCCGGATTTA
ACCAAGCCCTTTGAGCTGTTCGTTGATGAAAAA
CAGGGATATGCAAAAGGAGTATTAACCCAAAAG
TTAGGCCCGTGGCGTCGCCCTGTTGCTTACTTG
AGTAAAAAATTGGATCCTGTCGCAGCAGGATGG
CCACCGTGCTTGCGTATGGTCGCGGCAATTGCC
GTTTTGACAAAGGATGCAGGTAAGTTGACGATG
GGTCAACCCTTACGTATCTTGGCTCCACATGCT
GTAGAAGCGTTAGTAAAGCAGCCCCCAGACCGC
TGGCTTTCTAATGCGCGCATGACCCACTATCAG
GCGCTTCTGCTTGATACGGATCGTGTACAATTT
GGACCAGTTGTAGCTTTGAATCCAGCTACTTTG
CTTCCCCTTCCAGAAGAAGGACTTCAGCACAAT
TGTTTAGATATTCTGGCCGAGGCACATGGGACG
CGCCCTGATTTGACGGATCAGCCACTGCCTGAT
GCCGACCATACATGGTATACTGGCGGCAGTAGT
CTTCTTCAAGAGGGGCAACGCAAGGCGGGAGCA
GCCGTCACTACGGAGACCGAAGTTATCTGGGCC
AAAGCGTTACCCGCGGGAACATCCGCGCAACGT
GCACAGTTAATCGCTCTGACACAGGCCCTGAAG
ATGGCAGAGGGCAAAAAGTTGAATGTCTACACC
AACTCACGTTATGCTTTTGCAACAGCGCATGAA
CATGGCGAAATTTACCGCCGCCGTGGTCTGCTG
ACTAGTGAGGGTAAGGAAATTAAAAATAAAGAT
GAGATTCTTGCGTTGTTAAAAGCTTTATTCTTA
CCAAAACGCCTTTCGATCATTCATTGCCCGGGG
CATCAAAAGGGTCACTCAGCGGAGGCTCGTGGA
AACCGTATGGCGGACCAAGCTGCCCGTAAGGCG
GCGATCACAGAGACCCCGGATACATCAACGCTG
TTGATCGAAAACAGCTCTCCCTACACTAGCGAG
CATTTT
731 MMLV-II MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQA
Q68R/Q79R/L82Y/ WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
L99R/L280I/E282D/ MSREARLGIKPHIRRLYDQGILVPCQSPWNTPL
Q299E/T306K/V433R/ RPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN
I593E PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS
QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT
LFDEALHRDLADFRIQHPDLILLQYVDDLLLAA
TSELDCQQGTRALLQTLGNLGYRASAKKAQICQ
KQVKYLGYLLKEGQRWITDARKETVMGQPTPKT
PRELREFLGKAGFCRLWIPGFAEMAAPLYPLTK
TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDL
TKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL
SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
GQPLRILAPHAVEALVKQPPDRWLSNARMTHYQ
ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
CLDILAEAHGTRPDLTDQPLPDADHTWYTGGSS
LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHE
HGEIYRRRGLLTSEGKEIKNKDEILALLKALFL
PKRLSIIHCPGHQKGHSAEARGNRMADQAARKA
AITETPDTSTLLIENSSPYTSEHF
For the standard two-step procedure, RTases (1 μL, 620 nM) were added to a reaction mixture containing RNA (20 ng), dNTPs (100 μM), oligo dT primer (5 ng/uL) or both random hexamers and oligo dT primers (5 ng/uL each), first strand synthesis buffer (1×, 50 mM potassium acetate, 20 mM tris-acetate, pH 7.9, 10 mM magnesium acetate, 0.6 M trehalose 100 μg/ml BSA, and 10 mM DTT), and SuperaseIN (0.17 U/μL) in a 20 μL volume. The reaction proceeded at 50 or 65° C. for 15 minutes, followed by 80° C. for 10 minutes.
The subsequent cDNA synthesized by the RTase mutants in this disclosure were quantified by qPCR amplification using an assay that identified the SFRS9 gene in human cells. The assay master mix was a composition of Integrated DNA Technologies PrimeTime© Gene Expression Master Mix (GEM, 1×), SFRS9 primer set (500 nM, Table 3) and SFRS9 probe (250 nM, Table 3). The assay master mix and synthesized cDNA were mixed at a 10:1 ratio for a final volume of 20 μL. The reaction proceeded on a qPCR (QuantStudio7 Flex) using the following method: 95° C. hold for 3 minutes, followed by 95° C. for 15 seconds and 60° C. for one minute for 40 cycles. The reactions were analyzed and reported by Ct value (Tables 23-25). All mutant variants of MMLV RTase showed an increase in the overall activity compared to the base construct and three mutant variants of MMLV RTase showed noteworthy activity compared to the others, Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/V433N/I593W; Q68R/Q79R/L99R/E282D/Q299E/T306K/V433R/I593E; and Q68R/Q79R/L83Y/L99R/L280I/E282D/Q299E/T306K/V433R/I593E.
TABLE 23
Two-Step cDNA Synthesis by MMLV-RT mutants using oligo dT priming. The data was
generated via qPCR human normalizer assay and data is reported by Ct value.
RT
Temperature Ct Ct Standard
MMLV-RT Variant (° C.) Mean Deviation
MMLV-II 50 24.873 0.043
65 35.817 0.630
MMLV-II Q68R/Q79R/L99R/E282D 50 24.932 0.058
65 36.668 0.614
MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 50 24.750 0.036
65 35.782 1.366
MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433N/I593W 50 24.586 0.035
65 35.819 0.284
MMLV-II 50 24.638 0.028
Q68R/Q79R/L99R/E282D/L280EQ299E/V433N/I593W 65 34.319 0.343
MMLV-II 50 24.681 0.019
Q68R/Q79R/L82Y/L99R/E282D/L280I/Q299E/V433N/I593W 65 33.184 0.021
TABLE 24
Two-Step cDNA Synthesis by MMLV-RT mutants using oligo dT priming. The data was
generated via qPCR human normalizer assay and data is reported by Ct value.
RT
Temperature Ct Ct Standard
MMLV-RT Variant (° C.) Mean Deviation
MMLV-II 50 24.887 0.041
65 32.730 0.053
MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 50 25.061 0.126
65 27.898 0.070
MMLV-II 50 24.849 0.101
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433N/I593W 65 26.607 0.077
MMLV-II 50 25.110 0.154
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/V433N/I593W 65 25.701 0.062
MMLV-II 50 24.990 0.088
Q68R/Q79R/L99R/E282D/Q299E/T306K/V433R/I593E 65 25.929 0.114
MMLV-II 50 25.133 0.114
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433R 65 27.032 0.141
I593E
MMLV-II 50 24.817 0.122
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/ 65 25.721 0.187
V433R/I593E
TABLE 25
Two-Step cDNA Synthesis by MMLV-RT mutants using random priming. The data was
generated via qPCR human normalizer assay and data is reported by Ct value.
RT
Temperature Ct Ct Standard
MMLV-RT Variant (° C.) Mean Deviation
MMLV-II 50 25.048 0.075
65 32.563 0.156
MMLV-II Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 50 25.002 0.027
65 28.062 0.106
MMLV-II 50 25.016 0.179
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433N/ 65 26.724 0.040
I593W
MMLV-II 50 24.973 0.021
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/ 65 25.732 0.061
V433N/I593W
MMLV-II 50 24.982 0.030
Q68R/Q79R/L99R/E282D/Q299E/T306K/V433R/I593E 65 26.006 0.020
MMLV-II 50 25.078 0.065
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/V433R/I593E 65 27.080 0.122
MMLV-II 50 25.074 0.094
Q68R/Q79R/L82Y/L99R/L280I/E282D/Q299E/T306K/ 65 25.784 0.100
V433R/I593E
Example 7. Reverse Transcriptase Mutant Evaluation by Gene Specific Priming This example demonstrates the procedure used to evaluate each mutant RTase's ability to synthesize cDNA from purified RNA ultramers (Integrated DNA Technologies) compared to the base construct of MMLV RTase. The mutant MMLV RTases were tested by a one-step addition of the RTase in GEM as described in Example 5. The reactions were analyzed and reported by Ct value (Table 26). Twelve mutant variants of MMLV RTase showed an increase in the overall activity compared to the base construct, H77A, D83E, D83R, Y271E, Q299E, G308E, F396A, V433R, I593E, I597A, and I597R.
TABLE 26
One-Step cDNA Synthesis by MMLV-RT single mutants by gene
specific priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 29.065 0.277
MMLV-II D209A 29.583 0.166
MMLV-II D209E 28.900 0.088
MMLV-II D209R 29.266 0.068
MMLV-II D83 A 29.588 0.082
MMLV-II D83E 28.499 0.087
MMLV-II D83R 28.724 0.087
MMLV-II E201A 30.692 0.173
MMLV-II E201D 29.130 0.157
MMLV-II E201R 29.333 0.141
MMLV-II E367A 31.153 0.021
MMLV-II E367D 31.070 0.187
MMLV-II E367R 34.221 0.475
MMLV-II E596A 29.150 0.121
MMLV-II E596D 30.494 0.081
MMLV-II E596R 31.787 0.227
MMLV-II F210A 33.639 0.196
MMLV-II F210E 34.982 0.065
MMLV-II F210R 37.201 1.986
MMLV-II F369A 29.055 0.063
MMLV-II F369E 36.856 0.508
MMLV-II F369R 36.149 0.308
MMLV-II G308A 30.226 0.170
MMLV-II G308E 28.772 0.121
MMLV-II G308R 40.000 0.000
MMLV-II G331A 30.412 0.137
MMLV-II G331E 31.321 0.160
MMLV-II G331R 31.340 0.020
MMLV-II G73A 30.741 0.125
MMLV-II G73E 34.319 0.369
MMLV-II G73R 29.721 0.061
MMLV-II H77A 28.581 0.070
MMLV-II H77E 29.475 0.107
MMLV-II H77R 29.726 0.120
MMLV-II I125A 29.812 0.043
MMLV-II I125E 30.712 0.147
MMLV-II I125R 30.324 0.012
MMLV-II I212A 29.586 0.086
MMLV-II I212E 29.459 0.073
MMLV-II I212R 29.037 0.092
MMLV-II I593A 30.560 0.101
MMLV-II I593E 27.779 0.056
MMLV-II I593R 29.268 0.012
MMLV-II I597A 28.983 0.024
MMLV-II I597E 29.583 0.143
MMLV-II I597R 28.671 0.103
MMLV-II K285A 32.375 0.158
MMLV-II K285E 37.065 0.044
MMLV-II K285R 30.564 0.075
MMLV-II K348A 34.241 0.516
MMLV-II K348E 34.533 0.432
MMLV-II K348R 29.703 0.225
MMLV-II L198A 31.900 0.054
MMLV-II L198E 34.193 0.167
MMLV-II L198R 30.819 0.077
MMLV-II L280A 35.724 0.175
MMLV-II L280E 40.000 0.000
MMLV-II L280R 40.000 0.000
MMLV-II L352A 28.936 0.043
MMLV-II L352E 30.177 0.059
MMLV-II L352R 29.371 0.063
MMLV-II L357A 38.802 1.694
MMLV-II L357E 40.000 0.000
MMLV-II L357R 40.000 0.000
MMLV-II L82A 31.245 0.035
MMLV-II L82E 31.384 0.122
MMLV-II L82R 29.682 0.116
MMLV-II N335A 29.668 0.086
MMLV-II N335E 29.113 0.058
MMLV-II N335R 32.323 5.429
MMLV-II P76A 29.463 0.123
MMLV-II P76E 30.030 0.163
MMLV-II P76R 29.443 0.028
MMLV-II Q213A 29.833 0.223
MMLV-II Q213E 29.677 0.196
MMLV-II Q213R 29.704 0.053
MMLV-II Q299A 31.314 0.200
MMLV-II Q299E 28.652 0.149
MMLV-II Q299R 31.711 0.062
MMLV-II Q654A 29.415 0.117
MMLV-II Q654E 30.523 0.057
MMLV-II Q654R 29.523 0.052
MMLV-II R205A 29.140 0.138
MMLV-II R205E 29.356 0.179
MMLV-II R205K 29.162 0.206
MMLV-II R211A 29.491 0.025
MMLV-II R211E 30.049 0.205
MMLV-II R211K 30.196 0.147
MMLV-II R311A 31.237 0.425
MMLV-II R311E 40.000 0.000
MMLV-II R311K 29.857 0.091
MMLV-II R389A 32.173 0.151
MMLV-II R389E 32.717 0.105
MMLV-II R389K 31.944 0.166
MMLV-II R650A 29.734 0.060
MMLV-II R650E 31.012 0.074
MMLV-II R650K 29.404 0.094
MMLV-II R657A 31.470 0.133
MMLV-II R657E 32.785 0.145
MMLV-II R657K 29.468 0.274
MMLV-II S67A 29.268 0.090
MMLV-II S67E 30.157 0.254
MMLV-II S67R 27.274 0.054
MMLV-II T328A 40.000 0.000
MMLV-II T328E 37.699 1.627
MMLV-II T328R 37.169 0.848
MMLV-II T332A 29.219 0.075
MMLV-II T332E 29.714 0.057
MMLV-II T332R 30.462 0.130
MMLV-II V129A 29.305 0.077
MMLV-II V129E 31.188 0.181
MMLV-II V129R 30.383 0.081
MMLV-II V433A 30.483 0.059
MMLV-II V433E 30.106 0.144
MMLV-II V433R 29.297 0.457
MMLV-II V476A 31.295 0.244
MMLV-II V476E 34.664 0.364
MMLV-II V476R 31.223 0.166
MMLV-II Y271A 30.854 0.086
MMLV-II Y271E 28.620 0.068
MMLV-II Y271R 33.280 0.258
MMLV-IV 26.368 0.057
Example 8. Further Stacking of Reverse Transcriptase Mutants with Enhanced Activity This example demonstrates the procedure used to stack the enhanced mutants found in Examples 6 and 7 to further improve the MMLV RTase's ability to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) compared to the base construct and previously found mutant MMLV RTase containing the following mutations: Q68R/Q79R/L99R/E282D. The stacked mutant MMLV RTases were cloned, overexpressed and purified as described in Examples 1 and 2 and tested as described in Examples 6 and 7. Both the two- and one-step reactions were analyzed and reported by Ct value (Tables 27-29). Six of the eight stacked mutant variants of MMLV RTase increased the overall activity and thermostability compared to the base construct, Q68R/Q79R/L99R/E282D/V433R, Q68R/Q79R/L99R/E282D/1593E, Q68R/Q79R/L99R/E282D/Q299E, Q68R/Q79R/L99R/E282D/T332E, Q68R/L82R/L99R/E282D and Q68R/Q79R/L82R/L99R/E282D. Sub sequentially, four of those six stacked mutant variants of MMLV RTase increased the overall activity and thermostability compared to the previously identified mutant RTase (Q68R/Q79R/L99R/E282D), Q68R/Q79R/L99R/E282D/1593E, Q68R/Q79R/L99R/E282D/Q299E, Q68R/L82R/L99R/E282D and Q68R/Q79R/L82R/L99R/E282D.
Following these stacked mutant variants, MMLV RTase mutations were stacked further to improve the ability of MMLV RTase to synthesize cDNA from purified total RNA (DNased, isolated from HeLa cells) as compared to the MMLV RTase base construct (RNase H minus construct). Eight MMLV RTase sextuple or more mutant variants were cloned as described in Example 1 and overexpressed and purified as in Example 5.
MMLV RTase base construct and MMLV RTase mutant variants evaluated as described in Example 3. Temperatures were adjusted for both two-step and one-step reactions to 42/55 and 50/60° C., respectively. The two-step first strand synthesis buffer was modified from 50 mM Tris-hydrochloride, pH 8.3, 75 mM potassium chloride, 3 mM magnesium chloride and 10 mM DTT to 50 mM potassium acetate, 20 mM Tris-acetate, pH 7.0, 10 mM magnesium acetate, 100 pg/ml bovine serum albumin and 10 mM DTT. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (Tables 27-29).
Four of the eleven MMLV RTase sextuple or more mutant variants were found to exhibit increased overall activity and thermostability as compared to the other MMLV RTase stacked mutant variants, and almost all of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The four MMLV RTase mutant variants that were found to exhibit the highest overall activity were Q68R/Q79R/L99R/E282D/Q299E/V433R/V593E, Q68R2Q79R/L82R/L99R/E282D/Q299E/V433R/M593E, Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/593E, and Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E.
TABLE 27
Two-Step cDNA Synthesis by MMLV-RT stacked mutants using
oligo dT priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 37.388 0.396
MMLV-II Q68R/Q79R/L99R/E282D/V433R 29.215 0.113
MMLV-II Q68R/Q79R/L99R/E282D/I593E 33.563 0.118
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 31.902 0.169
MMLV-II Q68R/Q79R/L99R/E282D/T332E 33.988 0.108
MMLV-II Q68R/Q79R/L99R/L280R 40.000 0.000
MMLV-II Q68R/Q79R/L99R/L280R/E282D 40.000 0.000
MMLV-II Q68R/L82R/L99R/E282D 39.259 1.047
MMLV-II Q68R/Q79R/L82R/L99R/E282D 30.623 0.076
MMLV-IV 25.880 0.023
TABLE 28
Two-Step cDNA Synthesis by MMLV-RT stacked mutants using
random priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 36.638 1.014
MMLV-II Q68R/Q79R/L99R/E282D/V433R 40.000 0.000
MMLV-II Q68R/Q79R/L99R/E282D/I593E 32.331 0.111
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 30.430 0.154
MMLV-II Q68R/Q79R/L99R/E282D/T332E 33.720 0.266
MMLV-II Q68R/Q79R/L99R/L280R 40.000 0.000
MMLV-II Q68R/Q79R/L99R/L280R/E282D 40.000 0.000
MMLV-II Q68R/L82R/L99R/E282D 35.325 0.422
MMLV-II Q68R/Q79R/L82R/L99R/E282D 31.928 0.177
MMLV-IV 25.840 0.049
TABLE 29
One-Step cDNA Synthesis by MMLV-RT stacked mutants by gene
specific priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 33.027 0.048
MMLV-II Q68R/Q79R/L99R/E282D/V433R 29.937 0.040
MMLV-II Q68R/Q79R/L99R/E282D/I593E 28.724 0.081
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 29.341 0.022
MMLV-II Q68R/Q79R/L99R/E282D/T332E 30.330 0.036
MMLV-II Q68R/Q79R/L99R/L280R 40.000 0.000
MMLV-II Q68R/Q79R/L99R/L280R/E282D 40.000 0.000
MMLV-II Q68R/L82R/L99R/E282D 30.559 0.045
MMLV-II Q68R/Q79R/L82R/L99R/E282D 30.097 0.033
MMLV-IV 28.975 0.012
a. Evaluation of Ability of Purified MMLV RTase Mutant Variants to Synthesize DNA Over a Wide Range of Temperatures
MMLV RTase base construct MMLV RTase mutant variants evaluated as described in Example 5. Oligo-dT or random hexamer priming conditions and reaction temperatures were adjusted for the two-step reactions and RTase concentration was normalized to 31 nM. The two-step reactions for MMLV RTase base construct and MMLV RTase mutant variants were analyzed and reported by Ct output from the qPCR (see tables 25 and 26)
Five MMLV RTase mutants were found to exhibit high overall activity as compared to the MMLV RTase base construct over a wide range of temperatures, spanning from 37.0 to 51° C., regardless of which priming method used. All of the MMLV RTase stacked mutant variants exhibited increased overall activity and thermostability as compared to the MMLV RTase base construct. The five MMLV RTase mutant variants that were found to exhibit the highest overall activity at a wide range of temperatures were Q68R/Q79R/L99R/E282D, Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E, Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E, Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E and Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E
TABLE 30
Two-Step cDNA synthesis by MMLV RT quadruple and more mutants
by Oligo-dT priming. Data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Temperature of Ct Ct
MMLV RT Mutant Reaction (° C.) Mean SD
MMLV-II 37.0 26.340 0.033
MMLV-II 37.8 26.130 0.061
MMLV-II 39.5 25.830 0.014
MMLV-II 42.0 25.753 0.041
MMLV-II 45.2 25.632 0.077
MMLV-II 47.8 25.935 0.026
MMLV-II 49.2 26.478 0.042
MMLV-II 50.0 29.461 0.120
MMLV-II 51.0 29.430 0.098
MMLV-II 51.9 31.123 0.066
MMLV-II 53.8 33.632 0.073
MMLV-II 56.5 36.499 0.385
MMLV-II 59.9 37.158 0.427
MMLV-II 62.6 37.464 0.440
MMLV-II 64.2 37.082 0.022
MMLV-II 65.0 37.518 0.370
MMLV-II Q68R/Q79R/L99R/E282D 37.0 25.688 0.031
MMLV-II Q68R/Q79R/L99R/E282D 37.8 25.734 0.032
MMLV-II Q68R/Q79R/L99R/E282D 39.5 25.613 0.040
MMLV-II Q68R/Q79R/L99R/E282D 42.0 25.528 0.032
MMLV-II Q68R/Q79R/L99R/E282D 45.2 25.525 0.029
MMLV-II Q68R/Q79R/L99R/E282D 47.8 25.471 0.105
MMLV-II Q68R/Q79R/L99R/E282D 49.2 25.491 0.047
MMLV-II Q68R/Q79R/L99R/E282D 50.0 25.608 0.061
MMLV-II Q68R/Q79R/L99R/E282D 51.0 25.679 0.006
MMLV-II Q68R/Q79R/L99R/E282D 51.9 25.969 0.032
MMLV-II Q68R/Q79R/L99R/E282D 53.8 27.251 0.053
MMLV-II Q68R/Q79R/L99R/E282D 56.5 33.619 0.195
MMLV-II Q68R/Q79R/L99R/E282D 59.9 36.635 0.059
MMLV-II Q68R/Q79R/L99R/E282D 62.6 36.929 0.500
MMLV-II Q68R/Q79R/L99R/E282D 64.2 37.515 0.478
MMLV-II Q68R/Q79R/L99R/E282D 65.0 37.107 0.285
MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.0 26.133 0.054
MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.8 26.029 0.012
MMLV-II Q68R/Q79R/L99R/E282D/I593E 39.5 25.850 0.047
MMLV-II Q68R/Q79R/L99R/E282D/I593E 42.0 25.793 0.012
MMLV-II Q68R/Q79R/L99R/E282D/I593E 45.2 25.614 0.018
MMLV-II Q68R/Q79R/L99R/E282D/I593E 47.8 25.658 0.005
MMLV-II Q68R/Q79R/L99R/E282D/I593E 49.2 25.663 0.024
MMLV-II Q68R/Q79R/L99R/E282D/I593E 50.0 25.791 0.041
MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.0 25.877 0.067
MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.9 26.602 0.038
MMLV-II Q68R/Q79R/L99R/E282D/I593E 53.8 29.535 0.086
MMLV-II Q68R/Q79R/L99R/E282D/I593E 56.5 35.912 0.439
MMLV-II Q68R/Q79R/L99R/E282D/I593E 59.9 37.158 0.566
MMLV-II Q68R/Q79R/L99R/E282D/I593E 62.6 37.187 0.158
MMLV-II Q68R/Q79R/L99R/E282D/I593E 64.2 37.958 0.236
MMLV-II Q68R/Q79R/L99R/E282D/I593E 65.0 36.861 0.416
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.0 26.106 0.070
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.8 26.024 0.092
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 39.5 25.830 0.122
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 42.0 25.788 0.025
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 45.2 25.634 0.022
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 47.8 25.681 0.016
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 49.2 25.684 0.029
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 50.0 25.743 0.096
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.0 25.870 0.003
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.9 26.301 0.033
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 53.8 28.283 0.036
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 56.5 34.732 0.445
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 59.9 36.947 0.407
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 62.6 37.140 0.280
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 64.2 37.403 0.205
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 65.0 37.347 0.438
MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.0 25.961 0.170
MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.8 26.065 0.085
MMLV-II Q68R/Q79R/L82R/L99R/E282D 39.5 25.909 0.028
MMLV-II Q68R/Q79R/L82R/L99R/E282D 42.0 25.802 0.055
MMLV-II Q68R/Q79R/L82R/L99R/E282D 45.2 25.632 0.087
MMLV-II Q68R/Q79R/L82R/L99R/E282D 47.8 25.728 0.065
MMLV-II Q68R/Q79R/L82R/L99R/E282D 49.2 25.612 0.165
MMLV-II Q68R/Q79R/L82R/L99R/E282D 50.0 25.795 0.038
MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.0 25.830 0.009
MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.9 26.477 0.037
MMLV-II Q68R/Q79R/L82R/L99R/E282D 53.8 28.496 0.040
MMLV-II Q68R/Q79R/L82R/L99R/E282D 56.5 34.329 0.177
MMLV-II Q68R/Q79R/L82R/L99R/E282D 59.9 36.564 0.315
MMLV-II Q68R/Q79R/L82R/L99R/E282D 62.6 37.152 0.322
MMLV-II Q68R/Q79R/L82R/L99R/E282D 64.2 37.340 0.585
MMLV-II Q68R/Q79R/L82R/L99R/E282D 65.0 38.351 1.016
MMLV-II 37.0 25.853 0.057
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.8 25.898 0.016
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 39.5 25.716 0.093
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 42.0 25.669 0.064
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 45.2 25.643 0.056
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 47.8 25.680 0.016
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 49.2 25.663 0.057
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 50.0 25.708 0.045
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.0 25.557 0.025
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.9 26.015 0.125
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 53.8 27.812 0.048
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 56.5 34.073 0.217
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 59.9 36.512 0.168
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 62.6 37.182 0.167
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 64.2 37.239 0.291
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 65.0 36.573 0.232
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.0 25.789 0.075
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.8 25.784 0.103
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 39.5 25.714 0.025
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 42.0 25.713 0.027
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 45.2 25.690 0.030
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 47.8 25.662 0.026
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 49.2 25.713 0.021
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 50.0 25.551 0.092
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.0 25.561 0.107
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.9 25.975 0.125
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 53.8 27.556 0.023
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 56.5 33.934 0.249
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 59.9 36.473 0.285
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 62.6 37.411 0.377
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 64.2 37.656 0.478
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 65.0 37.950 1.451
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.0 25.788 0.028
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 37.8 25.680 0.229
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 39.5 25.794 0.051
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 42.0 25.415 0.270
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 45.2 25.631 0.047
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 47.8 25.672 0.027
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 49.2 25.792 0.045
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 50.0 25.759 0.022
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 51.0 25.852 0.015
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 51.9 26.425 0.033
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 53.8 29.964 0.023
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 56.5 36.532 0.113
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 59.9 38.246 0.608
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 62.6 37.333 0.446
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 64.2 37.223 0.212
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 65.0 36.930 0.527
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 37.0 25.863 0.014
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 37.8 25.649 0.036
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 39.5 25.573 0.057
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 42.0 25.453 0.023
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 45.2 25.447 0.083
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 47.8 25.413 0.061
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 49.2 25.542 0.035
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 50.0 25.567 0.060
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 51.0 25.741 0.093
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 51.9 26.231 0.225
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 53.8 28.556 0.142
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 56.5 35.202 0.208
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 59.9 36.991 0.419
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 62.6 37.168 0.463
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 64.2 37.670 0.410
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 65.0 37.680 0.273
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
TABLE 31
Two-Step cDNA synthesis by MMLV RT quadruple and more mutants
by Random priming. Data was generated via qPCR human normalizer
assay and data is reported by Ct value.
Temperature
of Reaction Ct Ct
MMLV RT Mutant (° C.) Mean SD
MMLV-II 37.0 26.365 0.066
MMLV-II 37.8 26.390 0.006
MMLV-II 39.5 25.939 0.016
MMLV-II 42.0 25.798 0.029
MMLV-II 45.2 25.849 0.064
MMLV-II 47.8 26.647 0.050
MMLV-II 49.2 28.326 0.028
MMLV-II 50.0 29.340 0.010
MMLV-II 51.0 30.684 0.099
MMLV-II 51.9 32.462 0.163
MMLV-II 53.8 33.855 0.307
MMLV-II 56.5 35.376 0.461
MMLV-II 59.9 36.098 0.481
MMLV-II 62.6 36.391 0.367
MMLV-II 64.2 36.442 0.547
MMLV-II 65.0 35.871 0.301
MMLV-II Q68R/Q79R/L99R/E282D 37.0 25.699 0.009
MMLV-II Q68R/Q79R/L99R/E282D 37.8 25.674 0.038
MMLV-II Q68R/Q79R/L99R/E282D 39.5 25.594 0.029
MMLV-II Q68R/Q79R/L99R/E282D 42.0 25.496 0.016
MMLV-II Q68R/Q79R/L99R/E282D 45.2 25.431 0.011
MMLV-II Q68R/Q79R/L99R/E282D 47.8 25.420 0.036
MMLV-II Q68R/Q79R/L99R/E282D 49.2 25.481 0.023
MMLV-II Q68R/Q79R/L99R/E282D 50.0 25.646 0.035
MMLV-II Q68R/Q79R/L99R/E282D 51.0 25.979 0.012
MMLV-II Q68R/Q79R/L99R/E282D 51.9 26.591 0.053
MMLV-II Q68R/Q79R/L99R/E282D 53.8 28.345 0.091
MMLV-II Q68R/Q79R/L99R/E282D 56.5 32.976 0.109
MMLV-II Q68R/Q79R/L99R/E282D 59.9 34.407 0.158
MMLV-II Q68R/Q79R/L99R/E282D 62.6 35.130 0.014
MMLV-II Q68R/Q79R/L99R/E282D 64.2 34.866 0.258
MMLV-II Q68R/Q79R/L99R/E282D 65.0 35.317 0.299
MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.0 26.079 0.036
MMLV-II Q68R/Q79R/L99R/E282D/I593E 37.8 25.951 0.015
MMLV-II Q68R/Q79R/L99R/E282D/I593E 39.5 25.801 0.055
MMLV-II Q68R/Q79R/L99R/E282D/I593E 42.0 25.602 0.087
MMLV-II Q68R/Q79R/L99R/E282D/I593E 45.2 25.424 0.038
MMLV-II Q68R/Q79R/L99R/E282D/I593E 47.8 25.520 0.011
MMLV-II Q68R/Q79R/L99R/E282D/I593E 49.2 25.674 0.046
MMLV-II Q68R/Q79R/L99R/E282D/I593E 50.0 25.922 0.015
MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.0 26.351 0.014
MMLV-II Q68R/Q79R/L99R/E282D/I593E 51.9 27.411 0.092
MMLV-II Q68R/Q79R/L99R/E282D/I593E 53.8 30.482 0.048
MMLV-II Q68R/Q79R/L99R/E282D/I593E 56.5 33.914 0.075
MMLV-II Q68R/Q79R/L99R/E282D/I593E 59.9 35.443 0.191
MMLV-II Q68R/Q79R/L99R/E282D/I593E 62.6 35.872 0.445
MMLV-II Q68R/Q79R/L99R/E282D/I593E 64.2 36.107 0.011
MMLV-II Q68R/Q79R/L99R/E282D/I593E 65.0 35.715 0.299
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.0 25.955 0.040
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 37.8 25.934 0.023
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 39.5 25.669 0.035
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 42.0 25.523 0.016
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 45.2 25.532 0.054
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 47.8 25.550 0.021
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 49.2 25.620 0.030
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 50.0 25.711 0.035
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.0 26.215 0.056
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 51.9 26.969 0.013
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 53.8 29.622 0.060
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 56.5 33.679 0.234
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 59.9 35.253 0.144
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 62.6 35.408 0.441
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 64.2 35.586 0.139
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 65.0 36.076 0.700
MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.0 25.884 0.012
MMLV-II Q68R/Q79R/L82R/L99R/E282D 37.8 25.833 0.009
MMLV-II Q68R/Q79R/L82R/L99R/E282D 39.5 25.684 0.077
MMLV-II Q68R/Q79R/L82R/L99R/E282D 42.0 25.553 0.026
MMLV-II Q68R/Q79R/L82R/L99R/E282D 45.2 25.471 0.043
MMLV-II Q68R/Q79R/L82R/L99R/E282D 47.8 25.491 0.085
MMLV-II Q68R/Q79R/L82R/L99R/E282D 49.2 25.646 0.014
MMLV-II Q68R/Q79R/L82R/L99R/E282D 50.0 25.765 0.039
MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.0 26.365 0.044
MMLV-II Q68R/Q79R/L82R/L99R/E282D 51.9 27.170 0.071
MMLV-II Q68R/Q79R/L82R/L99R/E282D 53.8 29.662 0.048
MMLV-II Q68R/Q79R/L82R/L99R/E282D 56.5 33.853 0.162
MMLV-II Q68R/Q79R/L82R/L99R/E282D 59.9 34.899 0.325
MMLV-II Q68R/Q79R/L82R/L99R/E282D 62.6 35.557 0.145
MMLV-II Q68R/Q79R/L82R/L99R/E282D 64.2 35.360 0.222
MMLV-II Q68R/Q79R/L82R/L99R/E282D 65.0 35.614 0.403
MMLV-II 37.0 25.706 0.031
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.8 25.757 0.101
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 39.5 25.435 0.036
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 42.0 25.417 0.025
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 45.2 25.425 0.023
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 47.8 25.401 0.049
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 49.2 25.467 0.009
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 50.0 25.516 0.056
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.0 25.880 0.039
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.9 26.348 0.064
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 53.8 28.506 0.018
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 56.5 32.812 0.242
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 59.9 34.123 0.163
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 62.6 35.108 0.027
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 64.2 34.796 0.171
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 65.0 34.999 0.064
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.0 25.711 0.080
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.8 25.916 0.224
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 39.5 25.665 0.052
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 42.0 25.527 0.016
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 45.2 25.504 0.065
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 47.8 25.437 0.070
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 49.2 25.555 0.065
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 50.0 25.571 0.028
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.0 25.854 0.029
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 51.9 26.259 0.057
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 53.8 28.329 0.053
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 56.5 32.962 0.212
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 59.9 34.072 0.446
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 62.6 34.931 0.205
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 64.2 34.626 0.169
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 65.0 35.085 0.230
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E
MMLV-II 37.0 25.940 0.130
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 37.8 25.793 0.129
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 39.5 25.599 0.015
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 42.0 25.504 0.016
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 45.2 25.602 0.041
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 47.8 25.604 0.058
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 49.2 25.665 0.007
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 50.0 25.821 0.068
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 51.0 26.315 0.047
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 51.9 27.036 0.059
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 53.8 31.004 0.089
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 56.5 33.765 0.274
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 59.9 34.656 0.209
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 62.6 35.561 0.468
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 64.2 35.877 0.154
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 65.0 35.659 0.477
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E
MMLV-II 37.0 25.780 0.046
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 37.8 25.652 0.026
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 39.5 25.641 0.037
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 42.0 25.507 0.005
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 45.2 25.484 0.067
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 47.8 25.438 0.027
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 49.2 25.534 0.022
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 50.0 25.755 0.085
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 51.0 25.981 0.027
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 51.9 26.242 0.052
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 53.8 29.146 0.069
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 56.5 33.138 0.159
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 59.9 34.551 0.152
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 62.6 35.186 0.322
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R
/I593E
MMLV-II 64.2 35.550 0.368
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
MMLV-II 65.0 35.459 0.295
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/
I593E
Example 9: Extension of Reverse Transcriptase Single Mutants The amino acid positions that enclosed the MMLV RTase single mutants identified in Examples 6 and 7 were further evaluated to include all possible amino acid substitutions at that position. The single mutants were cloned, overexpressed, and purified as described in Examples 1 and 2, and evaluated as described in Examples 6 and 7. The two-step and one-step reactions for MMLV RTase base construct and MMLV RTase double mutant variants were analyzed and reported by Ct output from the qPCR (Tables 32-34). Numerous single mutant MMLV RTase variants were found to exhibit an increase in the overall activity and thermostability as compared to the MMLV RTase base construct. The most prevalent among these were: L82F, L82K, L82T, L82Y, L280I, T332V, V433K, V433N, and I593W.
TABLE 32
Two-Step cDNA Synthesis by MMLV-RT single mutants using
Oligo-dT priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 40.000 0.000
MMLV-II I593A 40.000 0.000
MMLV-II I593C 37.874 0.991
MMLV-II I593D 40.000 0.000
MMLV-II I593E 40.000 0.000
MMLV-II I593F 40.000 0.000
MMLV-II I593G 39.748 0.356
MMLV-II I593H 39.502 0.704
MMLV-II I593K 40.000 0.000
MMLV-II I593L 38.994 1.423
MMLV-II I593M 39.383 0.873
MMLV-II I593N 40.000 0.000
MMLV-II I593P 40.000 0.000
MMLV-II I593Q 40.000 0.000
MMLV-II I593R 40.000 0.000
MMLV-II I593S 39.614 0.545
MMLV-II I593T 37.709 0.520
MMLV-II I593V 40.000 0.000
MMLV-II I593W 30.504 0.073
MMLV-II I593Y 40.000 0.000
MMLV-II L280A 40.000 0.000
MMLV-II L280C 40.000 0.000
MMLV-II L280D 40.000 0.000
MMLV-II L280E 40.000 0.000
MMLV-II L280F 40.000 0.000
MMLV-II L280G 40.000 0.000
MMLV-II L280H 40.000 0.000
MMLV-II L280I 30.951 0.076
MMLV-II L280K 40.000 0.000
MMLV-II L280M 40.000 0.000
MMLV-II L280N 39.727 0.386
MMLV-II L280P 40.000 0.000
MMLV-II L280Q 40.000 0.000
MMLV-II L280R 39.994 0.009
MMLV-II L280S 40.000 0.000
MMLV-II L280T 40.000 0.000
MMLV-II L280V 37.749 0.142
MMLV-II L280W 40.000 0.000
MMLV-II L280Y 40.000 0.000
MMLV-II L82A 40.000 0.000
MMLV-II L82C 39.565 0.615
MMLV-II L82D 40.000 0.000
MMLV-II L82E 40.000 0.000
MMLV-II L82F 39.347 0.924
MMLV-II L82G 40.000 0.000
MMLV-II L82H 40.000 0.000
MMLV-II L82I 40.000 0.000
MMLV-II L82K 37.136 0.593
MMLV-II L82M 38.649 1.260
MMLV-II L82N 40.000 0.000
MMLV-II L82P 40.000 0.000
MMLV-II L82Q 39.098 1.275
MMLV-II L82R 40.000 0.000
MMLV-II L82S 39.346 0.925
MMLV-II L82T 38.695 1.845
MMLV-II L82V 38.047 1.381
MMLV-II L82W 37.151 0.308
MMLV-II L82Y 35.014 0.421
MMLV-II Q299A 40.000 0.000
MMLV-II Q299C 40.000 0.000
MMLV-II Q299D 40.000 0.000
MMLV-II Q299E 39.061 1.328
MMLV-II Q299F 40.000 0.000
MMLV-II Q299G 40.000 0.000
MMLV-II Q299H 39.398 0.852
MMLV-II Q299I 39.183 1.155
MMLV-II Q299K 40.000 0.000
MMLV-II Q299L 39.474 0.743
MMLV-II Q299M 40.000 0.000
MMLV-II Q299N 40.000 0.000
MMLV-II Q299P 40.000 0.000
MMLV-II Q299R 40.000 0.000
MMLV-II Q299S 40.000 0.000
MMLV-II Q299T 40.000 0.000
MMLV-II Q299V 40.000 0.000
MMLV-II Q299W 40.000 0.000
MMLV-II Q299Y 40.000 0.000
MMLV-II T332A 39.087 1.291
MMLV-II T332C 38.956 1.476
MMLV-II T332D 40.000 0.000
MMLV-II T332E 39.554 0.631
MMLV-II T332F 40.000 0.000
MMLV-II T332G 37.321 2.009
MMLV-II T332H 39.215 1.110
MMLV-II T332I 39.344 0.927
MMLV-II T332K 40.000 0.000
MMLV-II T332L 40.000 0.000
MMLV-II T332M 37.775 1.632
MMLV-II T332N 37.326 0.834
MMLV-II T332P 40.000 0.000
MMLV-II T332Q 39.509 0.694
MMLV-II T332R 39.588 0.582
MMLV-II T332S 39.765 0.332
MMLV-II T332V 36.977 0.384
MMLV-II T332W 40.000 0.000
MMLV-II T332Y 40.000 0.000
MMLV-II V433A 40.000 0.000
MMLV-II V433C 37.504 0.682
MMLV-II V433D 40.000 0.000
MMLV-II V433E 35.189 0.336
MMLV-II V433F 39.379 0.878
MMLV-II V433G 39.482 0.732
MMLV-II V433H 40.000 0.000
MMLV-II V433I 39.781 0.310
MMLV-II V433K 35.770 0.623
MMLV-II V433L 39.015 0.744
MMLV-II V433M 39.119 1.247
MMLV-II V433N 33.981 0.185
MMLV-II V433P 40.000 0.000
MMLV-II V433Q 40.000 0.000
MMLV-II V433R 37.230 1.247
MMLV-II V433S 37.850 0.846
MMLV-II V433T 37.564 1.895
MMLV-II V433W 37.770 1.622
MMLV-II V433Y 40.000 0.000
MMLV-IV 26.102 0.033
TABLE 33
Two-Step cDNA Synthesis by MMLV-RT single mutants using
random priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Standard
MMLV-RT Variant Ct Mean Deviation
MMLV-II 40.000 0.000
MMLV-II I593A 40.000 0.000
MMLV-II I593C 40.000 0.000
MMLV-II I593D 39.992 0.012
MMLV-II I593E 40.000 0.000
MMLV-II I593F 39.189 1.147
MMLV-II I593G 40.000 0.000
MMLV-II I593H 40.000 0.000
MMLV-II I593K 40.000 0.000
MMLV-II I593L 40.000 0.000
MMLV-II I593M 40.000 0.000
MMLV-II I593N 40.000 0.000
MMLV-II I593P 40.000 0.000
MMLV-II I593Q 39.201 0.853
MMLV-II I593R 38.928 1.516
MMLV-II I593S 39.025 1.379
MMLV-II I593T 38.385 1.227
MMLV-II I593V 39.574 0.603
MMLV-II I593W 32.572 0.054
MMLV-II I593Y 40.000 0.000
MMLV-II L280A 40.000 0.000
MMLV-II L280C 40.000 0.000
MMLV-II L280D 40.000 0.000
MMLV-II L280E 40.000 0.000
MMLV-II L280F 40.000 0.000
MMLV-II L280G 40.000 0.000
MMLV-II L280H 40.000 0.000
MMLV-II L280I 34.152 0.276
MMLV-II L280K 40.000 0.000
MMLV-II L280M 39.973 0.038
MMLV-II L280N 40.000 0.000
MMLV-II L280P 40.000 0.000
MMLV-II L280Q 40.000 0.000
MMLV-II L280R 40.000 0.000
MMLV-II L280S 40.000 0.000
MMLV-II L280T 40.000 0.000
MMLV-II L280V 39.260 1.046
MMLV-II L280W 40.000 0.000
MMLV-II L280Y 40.000 0.000
MMLV-II L82A 40.000 0.000
MMLV-II L82C 40.000 0.000
MMLV-II L82D 40.000 0.000
MMLV-II L82E 39.672 0.463
MMLV-II L82F 36.854 0.708
MMLV-II L82G 40.000 0.000
MMLV-II L82H 37.705 0.557
MMLV-II L82I 39.231 1.087
MMLV-II L82K 39.437 0.443
MMLV-II L82M 40.000 0.000
MMLV-II L82N 40.000 0.000
MMLV-II L82P 40.000 0.000
MMLV-II L82Q 40.000 0.000
MMLV-II L82R 38.595 1.191
MMLV-II L82S 40.000 0.000
MMLV-II L82T 38.449 1.192
MMLV-II L82V 39.438 0.795
MMLV-II L82W 39.178 1.163
MMLV-II L82Y 36.758 0.962
MMLV-II Q299A 40.000 0.000
MMLV-II Q299C 40.000 0.000
MMLV-II Q299D 38.003 1.414
MMLV-II Q299E 39.338 0.936
MMLV-II Q299F 40.000 0.000
MMLV-II Q299G 40.000 0.000
MMLV-II Q299H 40.000 0.000
MMLV-II Q299I 39.850 0.212
MMLV-II Q299K 40.000 0.000
MMLV-II Q299L 40.000 0.000
MMLV-II Q299M 40.000 0.000
MMLV-II Q299N 40.000 0.000
MMLV-II Q299P 40.000 0.000
MMLV-II Q299R 40.000 0.000
MMLV-II Q299S 40.000 0.000
MMLV-II Q299T 40.000 0.000
MMLV-II Q299V 40.000 0.000
MMLV-II Q299W 40.000 0.000
MMLV-II Q299Y 40.000 0.000
MMLV-II T332A 39.814 0.264
MMLV-II T332C 40.000 0.000
MMLV-II T332D 40.000 0.000
MMLV-II T332E 40.000 0.000
MMLV-II T332F 40.000 0.000
MMLV-II T332G 38.897 1.560
MMLV-II T332H 40.000 0.000
MMLV-II T332I 40.000 0.000
MMLV-II T332K 40.000 0.000
MMLV-II T332L 38.169 2.589
MMLV-II T332M 37.410 1.906
MMLV-II T332N 38.983 1.362
MMLV-II T332P 39.046 1.350
MMLV-II T332Q 40.000 0.000
MMLV-II T332R 40.000 0.000
MMLV-II T332S 40.000 0.000
MMLV-II T332V 38.650 1.326
MMLV-II T332W 40.000 0.000
MMLV-II T332Y 40.000 0.000
MMLV-II V433A 40.000 0.000
MMLV-II V433C 37.605 0.184
MMLV-II V433D 40.000 0.000
MMLV-II V433E 34.693 0.193
MMLV-II V433F 40.000 0.000
MMLV-II V433G 40.000 0.000
MMLV-II V433H 40.000 0.000
MMLV-II V433I 39.792 0.294
MMLV-II V433K 35.725 0.464
MMLV-II V433L 40.000 0.000
MMLV-II V433M 40.000 0.000
MMLV-II V433N 34.604 0.554
MMLV-II V433P 40.000 0.000
MMLV-II V433Q 38.844 1.001
MMLV-II V433R 38.817 0.839
MMLV-II V433S 38.202 1.372
MMLV-II V433T 37.573 0.623
MMLV-II V433W 37.611 1.690
MMLV-II V433Y 40.000 0.000
MMLV-IV 26.053 0.098
TABLE 34
One-Step cDNA Synthesis by MMLV-RT single mutants by gene
specific priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Ct Ct Standard
MMLV-RT Variant Mean Deviation
MMLV-II 32.775 0.189
MMLV-II I593A 32.438 0.209
MMLV-II I593C 32.680 0.053
MMLV-II I593D 31.775 0.237
MMLV-II I593E 30.635 0.048
MMLV-II I593F 30.411 0.008
MMLV-II I593G 30.904 0.098
MMLV-II I593H 29.686 0.131
MMLV-II I593K 31.832 0.259
MMLV-II I593L 32.289 0.273
MMLV-II I593M 32.162 0.078
MMLV-II I593N 31.410 0.251
MMLV-II I593P 34.728 0.201
MMLV-II I593Q 31.609 0.032
MMLV-II I593R 31.144 0.133
MMLV-II I593S 30.548 0.247
MMLV-II I593T 29.572 0.236
MMLV-II I593V 30.673 0.142
MMLV-II I593W 28.179 0.092
MMLV-II I593Y 30.858 0.067
MMLV-II L280A 36.160 0.729
MMLV-II L280C 32.097 0.261
MMLV-II L280D 40.000 0.000
MMLV-II L280E 39.115 1.251
MMLV-II L280F 34.573 0.371
MMLV-II L280G 40.000 0.000
MMLV-II L280H 37.255 0.322
MMLV-II L280I 29.267 1.032
MMLV-II L280K 34.274 0.095
MMLV-II L280M 32.746 0.223
MMLV-II L280N 39.677 0.457
MMLV-II L280P 33.045 0.095
MMLV-II L280Q 39.190 1.145
MMLV-II L280R 40.000 0.000
MMLV-II L280S 40.000 0.000
MMLV-II L280T 37.074 0.325
MMLV-II L280V 30.461 0.052
MMLV-II L280W 40.000 0.000
MMLV-II L280Y 40.000 0.000
MMLV-II L82A 31.729 0.308
MMLV-II L82C 31.131 0.192
MMLV-II L82D 34.280 0.227
MMLV-II L82E 32.973 0.430
MMLV-II L82F 29.760 0.030
MMLV-II L82G 33.066 0.217
MMLV-II L82H 30.098 0.078
MMLV-II L82I 31.605 0.083
MMLV-II L82K 29.258 0.015
MMLV-II L82M 30.280 0.027
MMLV-II L82N 33.074 0.323
MMLV-II L82P 38.754 1.762
MMLV-II L82Q 32.001 0.164
MMLV-II L82R 30.208 0.128
MMLV-II L82S 31.841 0.231
MMLV-II L82T 28.908 0.044
MMLV-II L82V 29.533 0.057
MMLV-II L82W 29.580 0.056
MMLV-II L82Y 28.934 0.073
MMLV-II Q299A 31.113 0.138
MMLV-II Q299C 35.953 0.542
MMLV-II Q299D 32.292 0.080
MMLV-II Q299E 31.663 0.027
MMLV-II Q299F 36.143 0.317
MMLV-II Q299G 31.929 0.131
MMLV-II Q299H 32.387 0.133
MMLV-II Q299I 37.763 1.582
MMLV-II Q299K 32.326 0.096
MMLV-II Q299L 34.807 0.180
MMLV-II Q299M 32.514 0.375
MMLV-II Q299N 34.040 0.186
MMLV-II Q299P 39.460 0.764
MMLV-II Q299R 33.044 0.354
MMLV-II Q299S 33.438 0.256
MMLV-II Q299T 35.093 0.926
MMLV-II Q299V 35.114 1.045
MMLV-II Q299W 38.998 1.417
MMLV-II Q299Y 39.055 1.336
MMLV-II T332A 30.528 0.084
MMLV-II T332C 30.785 0.135
MMLV-II T332D 33.310 0.348
MMLV-II T332E 32.711 0.106
MMLV-II T332F 33.201 0.179
MMLV-II T332G 30.424 0.054
MMLV-II T332H 31.913 0.306
MMLV-II T332I 32.072 0.115
MMLV-II T332K 31.591 0.082
MMLV-II T332L 34.011 0.133
MMLV-II T332M 29.039 0.164
MMLV-II T332N 29.500 0.135
MMLV-II T332P 33.976 0.272
MMLV-II T332Q 31.599 0.041
MMLV-II T332R 32.950 0.130
MMLV-II T332S 31.003 0.341
MMLV-II T332V 29.835 0.061
MMLV-II T332W 35.431 0.099
MMLV-II T332Y 33.384 0.164
MMLV-II V433A 30.757 0.105
MMLV-II V433C 29.901 0.305
MMLV-II V433D 34.152 0.170
MMLV-II V433E 28.868 0.011
MMLV-II V433F 31.529 0.009
MMLV-II V433G 33.663 0.412
MMLV-II V433H 31.811 0.069
MMLV-II V433I 30.460 0.071
MMLV-II V433K 30.040 0.109
MMLV-II V433L 31.758 0.063
MMLV-II V433M 30.791 0.095
MMLV-II V433N 28.566 0.074
MMLV-II V433P 37.436 1.824
MMLV-II V433Q 30.586 0.104
MMLV-II V433R 30.773 0.080
MMLV-II V433S 29.768 0.074
MMLV-II V433T 29.096 0.107
MMLV-II V433W 29.130 0.064
MMLV-II V433Y 32.676 0.279
MMLV-IV 25.979 0.043
TABLE 35
Two-Step cDNA Synthesis by MMLV-RT stacked mutants using
oligo dT priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Temperature Ct Ct Standard
MMLV-RT Variant (° C.) Mean Deviation
MMLV-II 42 25.207 0.025
MMLV-II 55 28.180 0.022
MMLV-II 42 25.287 0.068
Q68R/Q79R/L99R/E282D 55 26.442 0.044
MMLV-II 42 25.344 0.065
Q68R/Q79R/L99R/E282D/V433R 55 26.586 0.077
MMLV-II 42 25.266 0.112
Q68R/Q79R/L99R/E282D/I593E 55 27.389 0.069
MMLV-II 42 25.357 0.087
Q68R/Q79R/L99R/E282D/Q299E 55 26.953 0.034
MMLV-II 42 25.394 0.011
Q68R/Q79R/L82R/L99R/E282D 55 27.171 0.028
MMLV-II 42 25.371 0.061
Q68R/Q79R/L99R/E282D/Q299E/ 55 26.689 0.068
I593E
MMLV-II 42 25.258 0.035
Q68R/Q79R/L82R/L99R/E282D/ 55 26.979 0.034
Q299E/I593E
MMLV-II 42 25.171 0.006
Q68R/Q79R/L99R/E282D/Q299E/ 55 26.299 0.025
V433R/I593E
MMLV-II 42 25.146 0.052
Q68R/Q79R/L82R/L99R/E282D/ 55 26.320 0.036
Q299E/V433R/I593E
MMLV-II 42 25.176 0.044
Q68R/Q79R/L82R/L99R/E282D/ 55 26.750 0.040
Q299E/T332E/I593E
MMLV-II 42 25.110 0.046
Q68R/Q79R/L82R/L99R/E282D/ 55 26.587 0.049
Q299E/T332E/V433R/I593E
MMLV-IV 42 25.184 0.025
MMLV-IV 55 25.153 0.037
SuperScript-IV 42 25.082 0.073
SuperScript-IV 55 25.080 0.047
TABLE 36
Two-Step cDNA Synthesis by MMLV-RT stacked mutants using random priming. The data
was generated via qPCR human normalizer assay and data is reported by Ct value.
Temperature Ct Ct Standard
MMLV-RT Variant (C) Mean Deviation
MMLV-II 42 25.264 0.019
MMLV-II 55 28.443 0.014
MMLV-II Q68R/Q79R/L99R/E282D 42 25.399 0.040
55 26.484 0.072
MMLV-II Q68R/Q79R/L99R/E282D/V433R 42 25.324 0.063
55 26.794 0.065
MMLV-II Q68R/Q79R/L99R/E282D/I593E 42 25.278 0.025
55 27.616 0.058
MMLV-II Q68R/Q79R/L99R/E282D/Q299E 42 25.281 0.079
55 27.148 0.025
MMLV-II Q68R/Q79R/L82R/L99R/E282D 42 25.279 0.053
55 27.243 0.008
MMLV-II Q68R/Q79R/L99R/E282D/Q299E/I593E 42 25.409 0.065
55 26.704 0.066
MMLV-II 42 25.581 0.062
Q68R/Q79R/L82R/L99R/E282D/Q299E/I593E 55 26.605 0.028
MMLV-II 42 25.355 0.158
Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E 55 26.305 0.066
MMLV-II 42 25.418 0.120
Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E 55 26.403 0.055
MMLV-II 42 25.374 0.115
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E 55 26.747 0.065
MMLV-II 42 25.426 0.082
Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/ 55 26.481 0.017
I593E
MMLV-IV 42 25.394 0.162
MMLV-IV 55 25.185 0.022
SuperScript-IV 42 25.299 0.132
SuperScript-IV 55 25.214 0.021
TABLE 37
One-Step cDNA Synthesis by MMLV-RT stacked mutants by gene
specific priming. The data was generated via qPCR human
normalizer assay and data is reported by Ct value.
Temperature Concentration Ct Ct Standard
MMLV-RT Variant (° C.) of RT (nM) Mean Deviation
MMLV-II 50 0.28 26.401 0.022
1.4 24.701 0.061
7.0 24.664 0.007
60 0.28 31.134 0.205
1.4 28.109 0.042
7.0 27.644 0.061
MMLV-II 50 0.28 25.171 0.046
Q68R/Q79R/L99R/ 1.4 24.440 0.037
E282D 7.0 24.406 0.010
60 0.28 28.848 0.114
1.4 25.905 0.066
7.0 25.618 0.057
MMLV-II 50 0.28 24.967 0.068
Q68R/Q79R/L99R/ 1.4 24.386 0.015
E282D/V433R 7.0 24.433 0.079
60 0.28 28.516 0.051
1.4 25.803 0.063
7.0 25.620 0.035
MMLV-II 50 0.28 24.660 0.053
Q68R/Q79R/L99R/ 1.4 24.377 0.028
E282D/I593E 7.0 24.355 0.021
60 0.28 27.488 0.074
1.4 25.413 0.049
7.0 25.209 0.136
MMLV-II 50 0.28 25.044 0.094
Q68R/Q79R/L99R/ 1.4 24.422 0.023
E282D/Q299E 7.0 24.528 0.055
60 0.28 28.818 0.137
1.4 25.953 0.082
7.0 25.754 0.098
MMLV-II 50 0.28 25.014 0.152
Q68R/Q79R/L82R/ 1.4 24.467 0.020
L99R/E282D 7.0 24.507 0.046
60 0.28 28.743 0.076
1.4 26.662 0.012
7.0 25.883 0.022
MMLV-II 50 0.28 24.771 0.027
Q68R/Q79R/L99R/ 1.4 24.501 0.008
E282D/Q299E/I593E 7.0 24.485 0.087
60 0.28 27.721 0.057
1.4 25.836 0.030
7.0 25.199 0.016
MMLV-II 50 0.28 24.777 0.029
Q68R/Q79R/L82R/ 1.4 24.432 0.033
L99R/E282D/Q299E/ 7.0 24.435 0.024
I593E 60 0.28 27.854 0.035
1.4 25.613 0.028
7.0 25.072 0.030
MMLV-II 50 0.28 24.550 0.003
Q68R/Q79R/L99R/ 1.4 24.333 0.033
E282D/Q299E/V433R/ 7.0 24.345 0.030
I593E 60 0.28 26.399 0.051
1.4 25.236 0.040
7.0 25.105 0.050
MMLV-II 50 0.28 24.562 0.047
Q68R/Q79R/L82R/ 1.4 24.350 0.039
L99R/E282D/Q299E/ 7.0 24.302 0.015
V433R/I593E 60 0.28 26.459 0.022
1.4 25.247 0.069
7.0 25.001 0.050
MMLV-II 50 0.28 24.614 0.047
Q68R/Q79R/L82R/ 1.4 24.420 0.051
L99R/E282D/Q299E/ 7.0 24.361 0.021
T332E/I593E 60 0.28 26.769 0.089
1.4 25.609 0.041
7.0 25.348 0.043
MMLV-II 50 0.28 24.594 0.075
Q68R/Q79R/L82R/ 1.4 24.402 0.045
L99R/E282D/Q299E/ 7.0 24.291 0.057
T332E/V433R/I593E 60 0.28 26.591 0.018
1.4 25.517 0.048
7.0 25.193 0.027
MMLV-IV 50 0.28 24.397 0.091
1.4 24.303 0.062
7.0 24.189 0.039
60 0.28 25.807 0.045
1.4 25.180 0.037
7.0 24.625 0.011
SuperScript-IV 50 0.28 24.743 0.049
1.4 24.213 0.017
7.0 24.008 0.036
60 0.28 26.124 0.103
1.4 24.681 0.070
7.0 24.180 0.082
TABLE 38
Sequences of quadruple or more mutant MMLV RTase variants.
SEQ ID NO: Construct Construct Sequence (AA)
686 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
V433R TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
687 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
I593E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
688 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
Q299E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYINSRYAFATAHIHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
689 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
T332E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGELFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
690 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q681/Q791/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/L280R RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWR
TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
691 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/L280R/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
E282D TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWR
TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
692 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/L82R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/E282D RLGIKPHIQRLRDQGILVPCQSPWNTPLRPVKKPG
TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
693 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG
E282D TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRQLREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHIHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
694 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
Q299E/I593E TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHEHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
695 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG
E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYTNSRYAFATAHEHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
696 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L99R/E282D/ RLGIKPHIRRLLDQGILVPCQSPWNTPLRPVKKPG
Q299E/V433R/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
697 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG
E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
V433R/I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
698 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG
E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
T332E/I593E PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGELFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
699 MMLV-II TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAE
Q68R/Q79R/ TGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREA
L82R/L99R/ RLGIKPHIRRLRDQGILVPCQSPWNTPLRPVKKPG
E282D/Q299E/ TNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP
T332E/V433R/ PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP
I593E EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF
RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL
TDARKETVMGQPTPKTPRELREFLGTAGFCRLWIP
GFAEMAAPLYPLTKTGELFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT
KDAGKLTMGQPLRILAPHAVEALVKQPPDRWLSNA
RMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG
LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTGG
SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
AQLIALTQALKMAEGKKLNVYINSRYAFATAHEHG
EIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL
SIIHCPGHQKGHSAEARGNRMADQAARKAAITETP
DTSTLLIENSSPYTSEHF
BIBLIOGRAPHY
- 1. Coffin et al., “The discovery of reverse transcriptase,” Ann. Rev. Virol. 3(1): 29-51 (2016).
- 2. Hogrefe et al., “Mutant reverse transcriptase and methods of use,” U.S. Pat. No. 9,783,791.
- 3. Kotewicz et al., “Cloned genes encoding reverse transcriptase lacking RNase H activity,” U.S. Pat. No. 5,405,776.
- 4. Kotewicz et al., “Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity,” Nucleic Acids Res. 16(1): 265-77 (1988).
- 5. Rogers et al., “Novel Reverse Transcriptases for Use in High Temperature Nucleic Acid Synthesis.” U.S. Patent Application Publication No. US 2015/0210989 A1.
